bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 3310: Riboflavin targets autofluorescent-positive breast cancer stem cells upon light exposure
Authors: Fouladdel Shamileh , Luther Tahra , Harvey Kaitlin , Martin-Trevino Rachel , Granger Jill , Azizi Ebrahim , Wicha Max S.
Published: July 2016

Most tumors are hierarchically organized and driven by a population of cells that display stem cell properties. These cancer stem cells (CSCs) have been characterized by in vitro assays such as sphere formation and in vivo by tumor initiation. In addition cell surface markers have been utilized to enrich for CSC populations. More recently a subtype of CSCs have been reported to display autofluorescence (AF), a property mediated by cellular uptake and concentration of riboflavin. In order to characterize the molecular heterogeneity of CSCs, we studied AF positive sorted single cells in different subtypes of breast cancer cell lines and also patient derived xenograft (PDX) breast tumor cells. Exposure of cell lines and PDXs to riboflavin significantly increased the percentage of AF+ cells 3-5 fold. These sorted AF+ cells were enriched for tumorsphere forming capacity in vitro. Flow cytometry analysis revealed partial overlap of AF+ cells with the previously characterized EMT-CSC phenotype CD44+/CD24- cells. We also utilized the microfluidic C1 and BioMark HD instruments to determine the expression patterns of 96 target genes using TaqMan assays in a multiplex RT-qPCR setting at single cell resolution. Single cell gene expression data showed a distinct signature in AF+ cells with high expression levels of MKI67, PCNA, ABCG2, BRCA1, Jag2, EZH2, and MMP9 genes. Since AF is mediated by specific cellular uptake and concentration of riboflavin, and the report that riboflavin is capable of generating cytotoxic free radicals upon light exposure, we determined whether this property could be exploited to selectively target CSCs. The AF+ cells were cultured in the presence and absence of riboflavin, exposed to visible light and analyzed by MTT cytotoxicity assay. Data revealed significant cytotoxicity of riboflavin on AF+ cells that were exposed to visible light. In conclusion, AF+ cells show distinct cancer stem cell features and are sensitive to light-activated riboflavin. These findings will help better understanding of tumor biology to identify new target therapies for treatment of cancer patients.

Citation Format: Shamileh Fouladdel, Tahra Luther, Kaitlin Harvey, Rachel Martin-Trevino, Jill Granger, Ebrahim Azizi, Max S. Wicha. Riboflavin targets autofluorescent-positive breast cancer stem cells upon light exposure. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3310.
Abstract 3314: Targeting Hsp90 affects stem cell signaling in triple negative breast cancer
Authors: Ramamoorthy Prabhu , Rangarajan Parthasarathy , Jensen Roy , Anant Shrikant
Published: July 2016

Background: Triple Negative Breast Cancer (TNBC) exhibit dismal survival rates due to their propensity to develop distant metastases. Heat shock protein 90 (Hsp90) is a molecular chaperone that aids in the folding and maturation of various proteins involved in breast cancer progression and resistance to therapy. Many studies have also suggested that breast cancer's ability to proliferate, progress, and spread is due to the presence of a rare subpopulation of cancer stem cells. The aim of this study is to investigate the efficacy of HSP90 inhibitors celastrol and triptolide (from the Chinese herb “Thunder God of Vine” (Tripterygium wilfordii) on stem cells as a novel therapy for TNBC.

Methods: We performed in vitro studies using the TNBC cell lines BT20 and MDA-MB-231. We performed mammosphere assays to determine self-renewal capacity. For in vivo, we injected BT20 cells into flanks of athymic nude mice and treated with celastrol and triptolide at 3 mg/Kg bw and 0.25 mg/Kg bw, respectively.

Results: Both celastrol and triptolide significantly suppressed mammosphere size and number. Furthermore, expression of breast cancer stem cell markers DCLK1, ALDH1 and CD133 were significantly reduced in the two cell lines following treatment. Flow cytometry also suggested a significant reduction in DCLK1+, ALDH+ and CD133+ cells. Recently, Notch signaling has been shown to be critical for self-renewal of cancer stem cells. In cells treated with either celastrol or triptolide, there was a significant reduction in activated Notch intracellullar domain (NICD), and its downstream target Hes-1. However, in cells where we ectopically overexpressed NICD, neither compound was as potent as control, vector transfected cells in reducing 2D cell proliferation or 3D mammosphere formation, suggesting the direct role for inhibiting Notch activation as a mechanism of action for the two compounds. Furthermore, inhibition of Notch signaling pathway by using Υ-secretase inhibitor DAPT shows further reduction in mammosphere formation and Notch and its downstream target gene. We confirmed these finding in vivo using BT20 tumor xenografts grown in athymic nude mice. There was a reduction in the size and weight of tumors in mice treated with celastrol or triptolide. Western blot data showed that there is a decrease in activated Notch-1 protein and stem cell marker, DCLK1+ and ALDH+ in celastrol and triptolide treated xenograft tissue.

Conclusion: Taken together these data suggest that both celastrol and triptolide affect cancer stem cells in TNBC, in part through inhibition of Notch signaling.

Citation Format: Prabhu Ramamoorthy, Parthasarathy Rangarajan, Roy Jensen, Shrikant Anant. Targeting Hsp90 affects stem cell signaling in triple negative breast cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3314.
Abstract 3315: Breast cancer stem cell induction by COX-2 via EP4/PI3K-AKT/NOTCH-WNT axis: EP4 as therapeutic target
Authors: Majumder Mousumi , Xin Xiping , Liu Ling , Tutunea-Fatan Elena , Rodriguez-Torres Mauricio , Vincent Krista , Deweyert Andrew , Postovit Lynne-Marie , Hess David , Lala Peeyush K.
Published: July 2016

Cancer stem-like cells (SLC) resist conventional therapies, necessitating searches for SLC-specific targets. We established that cyclo-oxygenase(COX)-2 expression promotes human breast cancer progression by activation of the prostaglandin(PG)E-2 receptor EP4. Present study revealed that COX-2 induces SLCs by EP4-mediated NOTCH and WNT up-regulation. EP4 antagonist (EP4A) treatment ablated SLCs both in vitro and in vivo. Ectopic COX-2 over-expression in MCF-7 and SKBR-3 human breast cancer cell lines (named MCF-7-COX-2 and SKBR-3-COX-2) resulted in aggressive phenotypes: increased migration/invasion/proliferation, EMT, elevated SLCs, evidenced by spheroid formation for successive generations, increased ALDH activity and co-expression of COX-2/SLC markers. These changes were reversed with COX-2 inhibitor or EP4A, indicating dependence on COX-2/EP4 activities. COX-2 overexpression or EP4 agonist treatment of COX-2 low cells caused up-regulation of NOTCH/WNT pathway genes, blocked with PI3K/AKT inhibitors. Supporting above findings, micro-array analysis

showed up-regulation of numerous SLC-regulatory and EMT-associated genes in MCF-7-COX-2 cells. MCF-7-COX-2 cells showed increased orthotopic tumorigenicity and spontaneous multi-organ metastases in NOD/SCID/IL-2Rγ-deficient mice for successive generations with limiting cell inocula. Orthotopic tumors showed significant up-regulation of VEGF-A/C/D, Vimentin and phospho-AKT, down-regulation of E-Cadherin and enrichment of SLC marker positive and spheroid forming cells. MCF-7-COX-2 cells also showed increased lung colonization in NOD/SCID/GUSB-null mice, an effect reversed with EP4 knockdown or EP4A treatment. COX-2, EP4 and ALDH1A expression in situ in human breast cancer tissues were highly correlated with one other, more marked in progressive stage of disease. High COX-2/EP4 expression was linked with poor survival. Thus EP4 represents a novel SLC-ablative target in human breast cancer. (Supported by a grant of the OICR to PKL and a TBCRU fellowship to MM)

Citation Format: Mousumi Majumder, Xiping Xin, Ling Liu, Elena Tutunea-Fatan, Mauricio Rodriguez-Torres, Krista Vincent, Andrew Deweyert, Lynne-Marie Postovit, David Hess, Peeyush K. Lala. Breast cancer stem cell induction by COX-2 via EP4/PI3K-AKT/NOTCH-WNT axis: EP4 as therapeutic target. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3315.
Abstract 3319: Characterization of the CD44+/CD49f+/CD24- subpopulation in basal-like DCIS cells
Authors: Duru Nadire , Gernapudi Ramkishore , Lo Pang-Kuo , Yao Yuan , Wolfson Benjamin , Zhang Yongshu , Zhou Qun
Published: July 2016

The molecular mechanisms responsible for Ductal Carcinoma In Situ (DCIS)-Invasive Ductal Carcinoma (IDC) transition have yet to be elucidated. Due to the lack of molecularly targeted therapies, basal-like DCIS has a high risk of recurrence and progression to invasive and metastatic cancers. In this study, using a human DCIS cell line model and a novel single-cell clonogenic approach, we have identified and characterized the aggressive clones derived from single cells with the CD49f+/CD44+/CD24- surface markers. We found that aggressive clones had enhanced self-renewal, migratory and invasive capacities. The aggressive clones had elevated ALDH activity, lower global DNA methylation and increased expression of stem cell related genes including OCT4 and SOX2. Further mechanistic studies showed that linc-ROR and miR-10b were overexpressed in aggressive clones and are key regulators of their self-renewal and invasive abilities. Finally, we confirmed our in vitro results in vivo, demonstrating that aggressive clones were capable of forming tumors in nude mice, whereas non-aggressive clones were not. We believe that characterization of aggressive clones within the heterogeneous CD49f+/CD44+/CD24- DCIS population and having linc-ROR and miR10b as additional markers to distinguish them from non-aggressive clones would provide the basis to develop new chemoprevention agents.

Citation Format: Nadire Duru, Ramkishore Gernapudi, Pang-Kuo Lo, Yuan Yao, Benjamin Wolfson, Yongshu Zhang, Qun Zhou. Characterization of the CD44+/CD49f+/CD24- subpopulation in basal-like DCIS cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3319.
Abstract 3324: Salinomycin possesses anti-tumor activity and inhibits breast cancer stem-like cells via inhibition of STAT3 activation in triple-negative breast cancer cells
Authors: Kim Ji Young , An Hyunsook , Oh Eunhye , Cho Youngkwan , Lee Nahyun , Seo Jae Hong
Published: July 2016

Cancer stem cells (CSCs) play important roles in the formation, growth and recurrence of tumors, particularly following therapeutic intervention. Triple-negative breast cancer (TNBC) is an aggressive tumor subtype with an enriched CD44+/CD24- stem-like population. Salinomycin has received recent attention for its ability to target breast cancer stem cells (BCSCs), but the mechanisms of action involved are not fully understood. The objective of the present study was to investigate the effect of salinomycin on cell death, migration, and invasion, as well as CSC-like properties in TNBC. Salinomycin significantly induced anoikis-sensitivity, accompanied by caspase-3 and caspase-8 activation and PARP cleavage, during anchorage-independent growth. Salinomycin treatment also caused a marked suppression of cell migration and invasion with concomitant downregulation of MMP-9 and MMP-2 mRNA levels. Notably, salinomycin inhibited the formation of mammospheres and effectively reduced the CD44+/CD24- stem-like population during anchorage-independent growth. These observations were associated with the inhibition of STAT3 phosphorylation (Tyr705). Furthermore, interleukin-6 (IL-6)-induced STAT3 activation was strongly suppressed by salinomycin challenge. TUNEL analysis of MDA-MB-231-derived xenografts revealed that salinomycin administration elicited a significant reduction in tumor growth with a marked downregulation of CD44, ALDH1 and phopspho-STAT3 levels, but seemingly without the induction of apoptosis. Our findings shed further light on the mechanisms responsible for salinomycin's effects on triple-negative breast cancer stem cells.

Citation Format: Ji Young Kim, Hyunsook An, Eunhye Oh, Youngkwan Cho, Nahyun Lee, Jae Hong Seo. Salinomycin possesses anti-tumor activity and inhibits breast cancer stem-like cells via inhibition of STAT3 activation in triple-negative breast cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3324.
Abstract 3335: The high cMyc expression characterizes a small cell subpopulation in ALK-positive anaplastic large cell lymphoma that carry cancer stem-like properties
Authors: Wu Chengsheng , Zhang Haifeng , Gupta Nidhi , Abdulraheem Alshareef , Wang Qian , Huang Yunghsin , Lai Raymond
Published: July 2016

We have previously identified two phenotypically distinct cell subpopulations in ALK+ anaplastic large cell lymphoma (ALK+ALCL) based on their differential response to a Sox2 reporter (SRR2), with reporter responsive (RR) cells being more tumorigenic and chemoresistant than reporter unresponsive (RU) cells. The regulation of the RU/RR dichotomy remains to be elusive. Our bioinformatics analysis of the SRR2 sequence suggests that cMyc may be an important factor. In support of this concept, we found a substantially higher expression of cMyc and more cMyc and Sox2-SRR2 probe bindings in RR cells than in RU cells. Inhibition of cMyc in RR cells significantly decreased SRR2 luciferase activity, and overexpression of cMyc in RU cells significantly increased SRR2 luciferase activity. Correlating with this, cMyc inhibition in RR cells significantly attenuated the clonogenicity and sensitized cells to doxorubicin; while overexpression of cMyc in RU cells conferred to the potentiated clonogenicity and chemoresistance. Importantly, we found cMyc not only maintains Sox2 protein expression, but also promotes Sox2 SRR2 probe binding and its transcriptional activity. Moreover, we found the highly active Wnt canonical pathway (WCP) contributes to the high expression of cMyc in RR cells. Intriguingly, we observed a positive regulatory loop in RR cells, in which Sox2 up-regulates Wnt2B, which can activate the WCP and cMyc. Furthermore, the significance of cMyc in this context was further highlighted by the fact that RU cells with stably transfected c-MYC are phenotypically similar with RR cells in terms of the elevated activation of WCP and increased tumorigenicity in vivo. Lastly, we found cMyc is heterogeneously expressed in ALK+ALCL tumor cells (n = 5) by immunohistochemcial staining, and it co-localizes with active β-catenin, a marker of active WCP, in tumor cells by immunofluorescence assay. In conclusion, cMyc is a key regulatory factor in the determination of RU/RR dichotomy, which is linked to tumorigenic potential and chemo-resistance in ALK+ALCL.

Citation Format: Chengsheng Wu, Haifeng Zhang, Nidhi Gupta, Alshareef Abdulraheem, Qian Wang, Yunghsin Huang, Raymond Lai. The high cMyc expression characterizes a small cell subpopulation in ALK-positive anaplastic large cell lymphoma that carry cancer stem-like properties. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3335.
Abstract 3337: Targeting acute myeloid leukemia via anti-IL1RAP antibodies
Authors: Jiang Ping , Liu Bob Y. , Huang Jen , Lu Jennifer , Roy Sharmili , Mishra Madhavi , Zhao Xiaoxian , Lin Jeffrey , Hsi Eric D. , Junutula Jagath R.
Published: July 2016

The current standard of care for Acute Myeloid Leukemia (AML) is largely ineffective in terms of achieving long term remission. This is mostly due to inability to remove all of the leukemic cells, and there is accumulating evidence to support the remaining leukemic cells are Leukemic Stem Cells (LSC) which are resistant to standard chemotherapy. We and others (Barreyro L. et. al. 2012) have discovered that IL-1 receptor accessory protein (IL1RAP) is overexpressed on LSC and their progenitors, but not on normal hematopoietic stem cells (HSC). Moreover, knockdown/knockout of IL1RAP expression greatly decreases growth/survival of AML cells. Thus, we have screened anti-IL1RAP therapeutic antibodies by applying FACS based analysis and Complement-Dependent Cytotoxicity (CDC) assays on both AML cell lines and primary patient samples. In a parallel effort, we also applied a functional IL-1β-induced NF-κB signaling assay to identify function-blocking anti-IL1RAP antibodies. Colony Formation Cell (CFC) assay results demonstrated that lead IL1RAP antibodies can significantly impede growth of both AML blast cells and leukemic stem cell-like cells. In contrast, normal HSCs are unaffected by these antibodies. Lead anti-IL1RAP antibodies also showed tumor growth inhibition in an established EOL-1 orthotopic tumor model. One major challenge of targeting IL1RAP by an antibody is that a large amount of IL1RAP protein is secreted into human serum, serving as a potential “sink” that inhibits IL1RAP antibody efficacy. We addressed this by developing a novel IL1RAP antibody that binds preferentially to the membrane-bound IL1RAP protein as compared to the secreted form. We will discuss different therapeutic development strategies to further advance the utilization of these lead anti-IL1RAP therapeutic antibodies for treating AML and other myeloid leukemic indications.

Citation Format: Ping Jiang, Bob Y. Liu, Jen Huang, Jennifer Lu, Sharmili Roy, Madhavi Mishra, Xiaoxian Zhao, Jeffrey Lin, Eric D. Hsi, Jagath R. Junutula. Targeting acute myeloid leukemia via anti-IL1RAP antibodies. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3337.
Abstract 3341: Microarray analysis of gastrointestinal stromal tumor-originated spheroids
Authors: Kikuchi Hirotoshi , Miyazaki Shinichiro , Murakami Tomohiro , Matsumoto Tomohiro , Ozaki Yusuke , Hiramatsu Yoshihiro , Kamiya Kinji , Ohta Manabu , Sakaguchi Takanori , Konno Hiroyuki
Published: July 2016

Background: Although the main cause of gastrointestinal stromal tumor (GIST) is gain-of-function mutations in the c-kit gene in the interstitial cells of Cajal, concomitant genetic or epigenetic changes other than c-kit are thought to occur during tumor progression. Although sequential therapy with tyrosine kinase inhibitors (TKIs), imatinib mesylate, sunitinib malate and regorafenib, is an effective and standard treatment strategy for recurrent or metastatic GISTs, it is difficult to cure recurrent GISTs with TKIs alone. It has been reported that viable cells are often seen in tumors resected in response to imatinib therapy and they may cause refractory tumors after interruption of imatinib in responding patients with advanced GISTs. Therefore targeting these persistent clones, which may have stem cell characteristics, can be a novel treatment strategy to cure recurrent or metastatic GISTs. In this study, we generated spheroids from resected GIST and performed microarray analysis to identify gene sets typically expressed in GIST-originated spheroid (GISTOS).

Methods: According to the previous report of cancer tissue originated spheroid (CTOS) (Kondo J et al. PNAS 108:6235-40, 2011), GISTOS was prepared from fresh samples of 6 primary gastric GISTs. Total RNAs were extracted from 6 GISTOS and their original tumors and utilized for cDNA microarray analysis. None of the patients had received imatinib therapy before surgery. Gene expression levels were compared between GISTOSs and their original tumors.

Results: Hierarchical clustering revealed two distinct clusters, and distinguished GISTOS from original tumors. Gene set enrichment analysis (GSEA) revealed that the gene sets CISPLATIN_RESISTSNCE, METASTASIS_STROMAs and TUMOR_INVASIVENESS_2D were upregulated and gene sets CELL_CYCLE_G1_S and MIR21_TARGETS were downregulated in GISTOSs compared with original tumors. These gene sets appear to reflect distinctive features of GISTOS that consists of enriched stem cell populations.

Conclusions: We generated GIST-originated spheroids. Microarray analysis revealed gene sets up-regulated and down-regulated in GISTOS. These gene sets appear to reflect distinctive features of GISTOS and may serve as novel molecular markers of stem cells in GIST.

Citation Format: Hirotoshi Kikuchi, Shinichiro Miyazaki, Tomohiro Murakami, Tomohiro Matsumoto, Yusuke Ozaki, Yoshihiro Hiramatsu, Kinji Kamiya, Manabu Ohta, Takanori Sakaguchi, Hiroyuki Konno. Microarray analysis of gastrointestinal stromal tumor-originated spheroids. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3341.
Abstract 3349: Differential expression of CD44 and CD24 markers discriminates the epithelioid from fibroblastoid subset in a sarcomatoid renal cell carcinoma cell line: evidence of the existence of cancer stem cells in both subsets
Authors: Hsieh Chin-Hsuan , Chuang Cheng-Keng , Pang See-Tung , Chien Samuel , Lin Paul , Liao Shuen-Kuei
Published: July 2016

The co-existence of epithelioid and fibroblastoid subsets in the human sarcomatoid renal carcinoma cell line, RCC52, has been demonstrated previously based on clonal studies, in which only the epithelioid subset was found to be tumorigenic in NOD/SCID mice. Since only few clonal isolates were used to analyze in that study, we argued that the tumorigenicity results might not be truly reflective of this tumor line. We therefore in this study determined to use cytofluorometrically sorted cells to re-evaluate certain biological parameters including xenotransplantation. Using two monoclonal antibodies to CD44 and CD24 markers, we succeeded in identification and isolation of the two populations, and showed the CD44bright/CD24dim and CD44bright/CD24bright phenotypes corresponding to the epithelioid and fibroblastoid subsets, respectively. At variance with the results of clonal studies, both cytofluorometrically sorted subsets displayed different but significant degrees of tumorigenicities, indicating that each subset harbored its own cancer stem cells (CSCs). In consistence with the tumorigenicity findings, both sorted subsets showed in vitro migratory/invasive potential with greater ability exhibited by the CD44bright/CD24bright subset which was associated with higher expression of MMP-7, -8 and TIMP-1 transcripts. On the other hand, the CD44bright/CD24dim subset was found to be associated with higher expression of MMP-2, -9 and TIMP-2 transcripts. Furthermore, both sorted subsets expressed the RCC stem cell marker CD105 in a small proportion of cells (4-5%), and were able to covert to PAX2+ clear cells in areas in the xenografts to a greater extent by the CD44bright/CD24dim subset, highlighting high plasticity of each subset in different ways. Collectively, our findings confirm the co-existence of two morphologically and phenotypically distinct subsets, and document the presence of CSCs in both subsets for the first time in sarcomatoid RCC. The CSCs/mCSCs in the two subsets should therefore be considered as emerging therapeutic targets in the design of future treatment strategies for this subtype of renal cancer.

Citation Format: Chin-Hsuan Hsieh, Cheng-Keng Chuang, See-Tung Pang, Samuel Chien, Paul Lin, Shuen-Kuei Liao. Differential expression of CD44 and CD24 markers discriminates the epithelioid from fibroblastoid subset in a sarcomatoid renal cell carcinoma cell line: evidence of the existence of cancer stem cells in both subsets. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3349.
Abstract 3358: Expression of ALDH1, Bmi-1 and CD24 in human oral squamous cell carcinoma and its relationship with clinical factors
Authors: Tamatani Tetsuya , Takamaru Natsumi , Fujisawa Kenji , Nagai Hirokazu , Miyamoto Youji
Published: July 2016

[Background] A cancer stem cell (cancer initiating cell, CSC) is considered capable of self -replication, self-differentiation, drug resistance, and immune evasion. Recently, CSC has become increasingly important in the treatment of malignant tumors. Cancer stem cells express specific molecules termed CSC marker, including aldehyde dehydrogenase (ALDH) 1, B lymphoma Mo-MLV insertion region 1 homolog (Bmi-1) and CD24, and their expression has been reported to be the potential prognostic values. However, the prognostic values of ALDH1, Bmi1, and CD24 expression in patients with oral cancer are less understood.

[Purpose] The aims of present study were to evaluate the expression of ALDH1, Bmi1, and CD24 in oral squamous cell carcinoma (OSCC) and to elucidate the relationships among the CSC marker expression, clinical stages, histological differentiation, the classification of invasion mode, cerebral lymph node metastasis and disease-free survival rate.

[Materials and Methods] Tissue specimens were obtains from 103 patients with OSCC after surgery or biopsy. Immunohistochemistry was used to assess ALDH1, Bmi1, and CD24 protein using at least 10% staining-positive cells as the definition of positive staining.

[Results] Immunohistochemical analysis of 103 cases showed that 31 cases (30%) expressed ALDH1. There was no relationship between ALDH1 expression and clinical stages or metastasis. However, there was significant association between ALDH1 expression and histological differentiation, or classification of invasion mode or disease-free survival rate. Otherwise, 39 cases (38%) cases of 103 OSCC patients expressed Bmi-1. There was significant association between Bmi-1 expression and invasion mode or disease-free survival rate. Sixty four cases (62%) expressed CD24. There was significant association between CD24 expression and histological differentiation.

[Conclusions] These findings suggested that the expression of ALDH1 and Bmi-1 as CSC markers in OSCC may be good marker indicating survival in patients with OSCC.

Citation Format: Tetsuya Tamatani, Natsumi Takamaru, Kenji Fujisawa, Hirokazu Nagai, Youji Miyamoto. Expression of ALDH1, Bmi-1 and CD24 in human oral squamous cell carcinoma and its relationship with clinical factors. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3358.
Abstract 3360: EB-virus latent membrane protein 1 potentiates the stemness of nasopharyngealcarcinoma via preferential activation of PI3K/AKT pathway by a positive feedback loop
Authors: Yang Chang-Fu , Yang Guang-Da , Huang Tie-Jun , Huang Jia-Ling , Qian Chao-Nan , Huang Bi-Jun
Published: July 2016

Our previous study reported that Epstein-Barr virus(EBV)-encoded latent membrane protein 1 (LMP1) could induce development of CD44+/High stem-like cells in nasopharyngeal carcinoma (NPC). However, the molecular mechanisms that underlie modulation of cancer stem cells (CSCs) in NPC remain unclear. Here, we show that LMP1 induced CSC-like properties through promotion of the expression of epithelial-mesenchymal transition (EMT)-like cellular markers and through alterations in differentiation markers. Furthermore, LMP1 activated and triggered PI3K/AKT pathway, which subsequently stimulated expression of CSC markers, development of side population (SP) and tumor sphere formation. This suggests that PI3K/AKT pathway plays an important role in the induction and maintenance of CSC properties in NPC. Similarly, PI3K/AKT pathway was also activated by phosphorylase in LMP1-induced CD44+/High cells. In addition, LMP1 greatly increased expression of miR-21 and down-regulated expression of the miR-21 target, PTEN. Overexpression of miR-21 by transfection of miR-21 mimics into LMP1-transformed cells led to phosphorylase-mediated activation of the PI3K/AKT pathway and induction of CSCs. On the contrary, phosphorylation of the PI3K/AKT pathway and the expression of CSC were reversed by a miR-21 inhibitor. The specific inhibitor (Ly294002) of PI3K/AKT pathway significantly decreased expression of miR-21 and CSC markers and up-regulated the expression of PTEN, which indicates that miR-21 and PTEN are the downstream effectors of PI3K/AKT and that expression of these two effectors are related to development of NPC CSCs. Taken together, our novel findings indicate that LMP1, PI3K/AKT, miR-21 and PTEN constitute a positive feedback loop and play a key role in LMP1-induced CSCs in NPC.

Citation Format: Chang-Fu Yang, Guang-Da Yang, Tie-Jun Huang, Jia-Ling Huang, Chao-Nan Qian, Bi-Jun Huang. EB-virus latent membrane protein 1 potentiates the stemness of nasopharyngealcarcinoma via preferential activation of PI3K/AKT pathway by a positive feedback loop. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3360.
Abstract 3362: Expression of CD44v9, a cancer stem cell marker, in human hepatocellular carcinoma
Authors: Wada Fumitaka
Published: July 2016

Background: CD44 is a cell-surface glycoprotein involved in cell-cell interactions, cell adhesion, and migration. This protein has recently been identified as one of the crucial cell surface markers for cancer stem cells (CSCs) in several types of tumor. It has recently been reported that CD44 variant 9 (CD44v9) interacted with the glutamate-cystine transporter xCT, thereby contributing to increased intracellular synthesis of glutathione (GSH), a strong scavenger of reactive oxygen species (ROS). CSCs having such mechanism are highly resistant to ROS (Cancer Cell 2011). Of note, the function of xCT is inhibited by sulfasalazine (Leukemia 2001), a commonly used drug for ulcerative colitis.

Aim: The aims of this study were to assess 1) the expression levels of CD44v9 and xCT in human HCC tissues and HCC cell lines, and 2) possible synergistic anti-tumor effect of cisplatin and sulfasalazine on HCC cells.

Methods: Thirty HCC tissues were subjected to immunohistochemistry for CD44v9 and xCT. The human HCC cell lines HAK-1A and HAK-1B were used. HAK-1B was an aggressive sister cell line derived from HAK-1A, a well-differentiated one (Hepatology 1993). The cells were incubated with cisplatin with or without sulfasalazine at concentrations of 0, 50, 100, and 200 μM under attached and sphere-forming conditions. Western blot analysis was employed to evaluate protein expression levels.

Results: Membranous localization of CD44v9 was found in 2 HCC tissues, that were moderately and poorly differentiated ones. In such cases, the standard isoform CD44s was also strongly expressed. Expression levels of xCT were ubiquitously demonstrated in all HCC cases studied; however, it was robustly upregulated in sphere-formed HAK-1B cells, in concert with the upregulation of CD44v9 in these cells. Sphere formation of HAK-1B was significantly inhibited by co-treatment with cisplatin and sulfasalazine in a dose-dependent manner.

Conclusions: The CD44v9-xCT system was demonstrated in less differentiated HCC tissues. Indeed, poorly differentiated HCC cells (HAK-1B) under sphere formation robustly expressed both CD44v9 and xCT, suggesting that CSC-like cells favored this system. To overcome the ROS resistance of CSCs, delivering sulfasalazine in combination with anti-cancer drugs may open a new avenue to a promising treatment of chemorefractory HCC.

Citation Format: Fumitaka Wada. Expression of CD44v9, a cancer stem cell marker, in human hepatocellular carcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3362.
Abstract 3363: Resistin promotes tumor angiogenesis and lymphangiogenesis through c-Src/PI3K/AKT signaling pathway in human chondrosarcoma cells
Authors: Chi Meng-Ju , Tsai Hsiao-Chi , Tang Chih-Hsin
Published: July 2016

Chondrosarcoma is a kind of commonly found bone malignant tumor. The development of distant metastasis often leads to a significant decline in overall survival, and there are no specific therapeutic methods for it until today. For tumors to metastasize, angiogenesis and lymphangiogenesis are both important steps in the early periods. Resistin was discovered as an adipocyte-secreting adipokine, which may play a critical role in modulating cancer pathogenesis. However, the role of resistin in the angiogenesis and lymphangiogenesis of human chondrosarcoma is largely unknown. In our lab, we found that the expression of resistin was higher in chondrosarcoma biopsy tissues than in normal cartilage. We also confirmed that treated cells with resistin increased VEGFA and VEGFC expression, promoting angiogenesis as well as lymphangiogenesis in human chondrosarcoma cells. Moreover, the inhibitors and siRNAs of c-Src, PI3K, and AKT reduced the resistin-increased cell angiogenesis and lymphangiogenesis. Our results indicate that resistin promotes chondrosarcoma angiogenesis and lymphangiogenesis by the increasing VEGFA and VEGFC expression through the activation of the c-Src/PI3K/AKT signaling pathway expression. Therefore, resistin may represent a potential novel molecular therapeutic target for chondrosarcoma therapeutic treatment.

Citation Format: Meng-Ju Chi, Hsiao-Chi Tsai, Chih-Hsin Tang. Resistin promotes tumor angiogenesis and lymphangiogenesis through c-Src/PI3K/AKT signaling pathway in human chondrosarcoma cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3363.
Abstract 3364: Adaptive mechanisms upon LRP1 repression: Impact on sphingosine-1-phosphate-mediated signalling and migration of brain endothelial cells
Authors: Vézina Amélie , Charfi Cyndia , Zgheib Alain , Annabi Borhane
Published: July 2016

Switches in the sphingolipid metabolism have recently been associated with oncogenic transformation, and a role for the low density lipoprotein receptor related protein (LRP1) in the sphingosine-1-phosphate (S1P) proangiogenic signalling inferred. Although S1P is a key determinant of blood-brain barrier (BBB) permeability, the interdependent signalling crosstalk with LRP1 in brain endothelial cells (EC) remains unclear. Here, we tested whether any adaptive LRP1/S1P interdependence mechanisms are required in a SV40-immortalized human brain microvascular EC (HBMEC) model which closely mimics the brain tumor endothelium phenotype. Transient LRP1 silencing and stable LRP1-/- HBMEC were generated and increased expression of CCAAT/enhancer binding protein β (C/EBPβ), a transcription factor which regulates the transcription of all five S1P receptors members, was observed upon stable but not transient LRP1 repression. Migration of EC isolated from Lrp1(EC)-/- mice and of stable LRP1-/- HBMEC became unresponsive to S1P, in part explained by altered ERK and p38 MAPK pathways, whereas it remained unaltered in transient in vitro LRP1 repression. Adaptive diminished expressions of S1P1, S1P3, and S1P5, and increased S1P2 were observed in stable LRP1-/- HBMEC and in brain EC isolated from Lrp1(EC)-/- mice. Finally, gene silencing strategies highlighted the need for S1P3 for proper S1P-mediated HBMEC migration. Collectively, our study highlights an unexpected adaptive transcriptional crosstalk between LRP1 and specific S1P receptors which accounts for S1P signalling in brain EC. Targeting of the LRP1/S1P signalling axis at the BBB may thus be considered in future therapeutic strategies.

Citation Format: Amélie Vézina, Cyndia Charfi, Alain Zgheib, Borhane Annabi. Adaptive mechanisms upon LRP1 repression: Impact on sphingosine-1-phosphate-mediated signalling and migration of brain endothelial cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3364.
Abstract 3366: NCL targeting impairs the progression of pancreatic ductal adenocarcinoma and promotes tumor vessel normalization through Ang-2 inhibition
Authors: Gilles Maud-Emmanuelle , Maione Federica , Cossutta Mélissande , Carpentier Gilles , Caruana Laure , Di Maria Silvia , Destouches Damien , Shchors Ksenya , Prochasson Christopher , Couvelard Anne , Courty José , Giraudo Enrico , Cascone Ilaria
Published: July 2016

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive tumor, mostly resistant to the standard treatments. NCL is overexpressed in cancers and its inhibition impairs tumor growth. Herein we described, that NCL was overexpressed in human specimens of PDAC, and low NCL staining patients had increased overall survival. Previously, we described a family of multivalent pseudopeptides binding to NCL and inhibiting tumour growth. Here, NCL antagonist N6L, strongly impaired tumor growth, liver metastasis formation and angiogenesis in an orthothopic mouse model of PDAC. N6L inhibited both human and mouse tumor cell proliferation and invasion. Proteome analysis of endothelial cell secreted proteins showed that NCL inhibition decreased Ang-2 levels and switched a pro-angiogenic signature. Importantly, Ang-2 levels were decreased in plasma of N6L-treated PDAC mice. The analysis of tumor vasculature revealed a strong increase of pericyte coverage and vessel perfusion in parallel to an inhibition of tumor hypoxia. As consequence of N6L-induced tumor vessel normalization, pre-treatment with N6L efficiently improved chemotherapeutic drug delivery and increased the anti-tumor properties of gemcitabine in PDAC mice.

In conclusion, NCL inhibition is a new anti-tumor therapeutic strategy that dually blocks tumor progression and normalizes tumor vessels improving the delivery and efficacy of chemotherapeutic drugs in PDAC cancers. Moreover, we identified Ang-2 as a potential target and suitable response biomarker for N6L treatment in PDAC.

Citation Format: Maud-Emmanuelle Gilles, Federica Maione, Mélissande Cossutta, Gilles Carpentier, Laure Caruana, Silvia Di Maria, Damien Destouches, Ksenya Shchors, Christopher Prochasson, Anne Couvelard, José Courty, Enrico Giraudo, Ilaria Cascone. NCL targeting impairs the progression of pancreatic ductal adenocarcinoma and promotes tumor vessel normalization through Ang-2 inhibition. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3366.
Abstract 3369: Tumor-microenvironment-dependent imprinting of endothelial cells in human colorectal carcinoma
Authors: Stürzl Michael , Liebl Andrea , Schellerer Vera S. , Schütz Manuela , Kölbel Patrick , Schaal Ute , Haep Lisa , Klein-Hitpass Ludger , Rau Timan T. , Dietel Barbara , Meniel Valerie , Clarke Alan R. , Merkel Susanne , Croner Roland S. , Hohenberger Werner , Naschberger Elisabeth
Published: July 2016

The tumor microenvironment (TME) and tumor cell heterogeneity contribute significantly to tumor pathogenesis. In contrast, potential stromal cell heterogeneity induced by the TME and its impact on human tumorigenesis has not been conclusively investigated. Therefore, pure tumor endothelial cells (TECs) from human colorectal carcinomas (CRCs) with differential TMEs (favorable Th1-TME and worse control-TME) were isolated and significantly different gene clusters reflecting the tumorigenic and angiogenic impact attributed to the respective TMEs were identified by transcriptome analysis. SPARCL1 - the most strongly upregulated gene in Th1-TME-derived TECs - was analyzed to prove this concept. SPARCL1 was expressed by endothelial cells (EC) in colon tissues, most prominently in mature normal vessels, similarly in CRC with Th1-TME and lost towards more aggressive and angiogenic control tumors. SPARCL1 was induced in confluent, quiescent EC in culture but absent or resolved in angiogenically active EC. SPARCL1 inhibited proliferation, migration and sprouting of cultivated EC in vitro. In human CRC tissues SPARCL1 expressing vessels were highly covered by mural cells and of larger vessel size (perimeter and area), indicting more progressed maturation. Similarly the vessels in the colon of wild type mice as compared to SPARCL1/Sc1 knockout mice exhibited a more mature vessel structure. In conclusion, exploiting cellular memory processes we demonstrated TME-dependent intratumoral TEC heterogeneity in CRC and identified SPARCL1 as novel EC-derived protein regulating vessel maturation and homeostasis.

Citation Format: Michael Stürzl, Andrea Liebl, Vera S. Schellerer, Manuela Schütz, Patrick Kölbel, Ute Schaal, Lisa Haep, Ludger Klein-Hitpass, Timan T. Rau, Barbara Dietel, Valerie Meniel, Alan R. Clarke, Susanne Merkel, Roland S. Croner, Werner Hohenberger, Elisabeth Naschberger. Tumor-microenvironment-dependent imprinting of endothelial cells in human colorectal carcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3369.
Abstract 3376: Molecular drivers of endothelial hypoxic adaption during angiogenesis deciphered using the Berg Interrogative Biology® platform
Authors: Walshe Tony E. , Bourdelais Justin , Akmaev Viatcheslav R. , Rodrigues Leonardo O. , Lao Socheata , Gesta Stephane , Kiebish Michael A. , Vishnudas Vivek K. , Sarangarajan Rangaprasad , Narain Niven R.
Published: July 2016

Angiogenesis is a key feature of tumor progression providing oxygen and nutrients required for tumor cell growth, hypoxia being a major driver of this phenomenon. A systems biology approach using the Berg Interrogative Biology® platform was implemented to identify drivers of the endothelial cell (EC) angiogenic response to hypoxia. To determine the proteomic profile of proliferating ECs and non-proliferating confluent ECs exposed to normoxia or hypoxia, a functional proteomic approach employing activity-based probes and phosphorylation analysis was utilized. Kinases and ATPases were labeled with ATP-binding domain enrichment probes and titanium dioxide enrichment of phosphopeptides was employed for capture of protein phosphorylation events. Phenotypic assays including proliferation rates, apoptosis, mitochondrial superoxide and ROS/NO signaling were included as a measure of EC phenotype. Comparative proteomics, kinase activity, phosphoproteomics and assay data were integrated using an AI based Bayesian Network Inference approach to investigate causal signaling networks in order to elucidate the complexity and dynamics of angiogenesis and more specifically, the role of hypoxia in driving intracellular signaling in response to changes in oxygen tension. High confidence causal networks identified novel proteins that modulate the EC hypoxic response, validation of which are in process. Previously characterized proteins that are responsive to hypoxia were also identified in the hypoxic, but not normoxic signaling networks, namely Aldoc, Rac1, mTor, Cav-1 and Bax. Endothelial proliferation rate is closely related to both hydrogen peroxide and nitric oxide signaling, as has previously been reported. Novel regulatory networks that determine these interactions were identified. Using the Berg Interrogative Biology® platform, we are deciphering both the effects of the hypoxic microenvironment, and the unique characteristics of proliferating ECs by applying integrated functional proteomic assays and a systems approach to determine global changes in intracellular signaling in response to hypoxia.

Citation Format: Tony E. Walshe, Justin Bourdelais, Viatcheslav R. Akmaev, Leonardo O. Rodrigues, Socheata Lao, Stephane Gesta, Michael A. Kiebish, Vivek K. Vishnudas, Rangaprasad Sarangarajan, Niven R. Narain. Molecular drivers of endothelial hypoxic adaption during angiogenesis deciphered using the Berg Interrogative Biology® platform. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3376.
Abstract 3379: Tumor-induced remote extracellular matrix network orientation steers angiogenesis
Authors: Danen Erik H.
Published: July 2016

Tumor angiogenesis promotes tumor growth and metastasis. Here, we use automated sequential microprinting of tumor and endothelial cells in extracellular matrix (ECM) scaffolds to study its mechanical aspects. Quantitative reflection microscopy shows that tumor spheroids induce radial orientation of the surrounding collagen fiber network up to a distance of five times their radius. Across a panel of ∼20 different human tumor cell lines, remote collagen orientation is correlated with local tumor cell migration behavior. Tumor induced collagen orientation requires contractility but is remarkably resistant to depletion of collagen-binding integrins. Microvascular endothelial cells undergo directional migration towards tumor spheroids once they are within the tumor-oriented collagen fiber network. Laser ablation experiments indicate that an intact physical connection of the oriented network with the tumor spheroid is required for mechanical sensing by the endothelial cells. Together our findings show that remote physical manipulation of the ECM network by the tumor steers angiogenesis.

Citation Format: Erik H. Danen. Tumor-induced remote extracellular matrix network orientation steers angiogenesis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3379.