bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 3851: Molecular profiling of aggressive breast cancer in a unique patient population from Kenya
Authors: Torrorey Rispah , Kerper Maggie , Hartland Emilia , Shi Zonggao , Prosperi Jenifer , Badve Sunil , Stack Sharon , Mining Simeon , Littlepage Laurie
Published: May 2015

Breast cancer rates of incidence and mortality vary significantly between different nations and racial groups. African nations have the highest breast cancer mortality rates in the world, even though the incidence rates are below those of many other nations. In Kenya, breast cancer tumors are often highly aggressive at presentation and occur at a significantly earlier age (as early as the teens and 20s), relative to North American women. In the United States, non-Hispanic white women have the highest incidence of breast cancer, but African-American women have the highest mortality. These striking racial disparities are due not only to inequities in screening and treatment but also to variations in the rates of aggressive breast cancer. Differences in disease progression suggest that aggressive breast cancer tumors may harbor components of a unique molecular signature that result in racial disparities. We aim to identify drivers of poor prognosis breast cancer growth by identifying molecular signatures with high prognostic value from tumor samples of patients with aggressive disease. We hypothesize that changes in the DNA, RNA, and post-translational protein regulation contribute to aggressive disease. To characterize the tumors from this patient population, we used samples from >100 Kenyan breast tumor tissue samples. We stained tissue microarray sections for clinical breast cancer markers including lymphocyte markers. Using DNA and RNA that we isolated from these patient-derived breast tumors, we are characterizing these tumors by analyzing them for gene expression, genome sequencing, proteomics, and pathology analysis coupled with bioinformatics to develop signatures of aggressive breast cancer growth and metastasis. Our data will be foundational in understanding how aggressive, lethal breast tumors of Kenyan breast cancer patients differ from less aggressive tumors and will enhance our ability to diagnose and eliminate outcome disparities in breast cancer patients.

Citation Format: Rispah Torrorey, Maggie Kerper, Emilia Hartland, Zonggao Shi, Jenifer Prosperi, Sunil Badve, Sharon Stack, Simeon Mining, Laurie Littlepage. Molecular profiling of aggressive breast cancer in a unique patient population from Kenya. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3851. doi:10.1158/1538-7445.AM2014-3851
Abstract 3856: Progesterone generates breast cancer stem cells through nuclear receptor-independent mechanisms in irradiated human mammary cell lines
Authors: Vares Guillaume , Cui Xing , Wang Bing , Nakajima Tetsuo , Nenoi Mitsuru
Published: May 2015

Epidemiological evidence has shown the existence of increased breast cancer risk following chest radiation exposure. There is strong evidence that steroid hormones influence radiosensitivity and breast cancer risk. Tumors may be initiated by a small subpopulation of cancer stem cells (CSCs). In order to evaluate whether the modulation of radiation-induced breast cancer risk by steroid hormones could involve CSCs, we measured the proportion of ALDH+ and CD44+/CD24- CSCs in irradiated breast cancer cell lines after progesterone and estrogen treatment. Progesterone stimulated the expansion of the CSC compartment both in progesterone receptor (PR)-positive breast cancer cells and in PR-negative normal cells. In MCF10A normal epithelial PR-negative cells, progesterone-treatment and irradiation triggered cancer and stemness-associated microRNA regulations (such as the down-regulation of miR-22 and miR-29c expression), which resulted in increased proportions of radiation-resistant tumor-initiating CSCs. Therefore progesterone might influence radiation-induced breast cancer risk by generating tumor-initiating breast CSCs through nuclear PR-independent mechanisms (such as membrane progesterone receptors - mPRs).

Citation Format: Guillaume Vares, Xing Cui, Bing Wang, Tetsuo Nakajima, Mitsuru Nenoi. Progesterone generates breast cancer stem cells through nuclear receptor-independent mechanisms in irradiated human mammary cell lines. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3856. doi:10.1158/1538-7445.AM2014-3856
Abstract 3858: Implications of the downregulation of stemness/reprogramming factor expression by ibuprofen and biguanides
Authors: Grenlin Laura , Tran Tuan-Anh , Lomahan Sarah , Ikegaki Naohiko , Tang Xao X.
Published: May 2015

Cancer stem cells (CSCs) are thought to have the capacity to renew indefinitely, to initiate tumor formation, and to give rise to multiple non-tumorigenic progenies via asymmetric cell division. As a result of this phenotypic drift, an established tumor would consist of a mixture of CSC and non-CSC. Based on the current view, the CSC population is likely responsible for distant metastases, drug resistance and recurrence. However, the process by which this cell population is generated in a tumor mass has continued to be unclear. Based on our previous study (PNAS Vol. 110: 6097-6102, 2013), this process may involve nuclear reprogramming, namely the elevated expression of stemness/reprogramming factors (SOX2, OCT4, NANOG, LIN28, KLF4, MYC/MYCN, and those with equivalent functions). If so, it is conceivable that destabilization of these proteins would prevent or substantially slow down the generation of CSC compartment(s) within a tumor mass. Recently, we have reported that anti-inflammatory Ibuprofen and anti-diabetic biguanides destabilize MYC and MYCN in neuroblastoma cells. In this study, we have investigated the effects of Ibuprofen and Phenformin on stemness phenotypes of cancer cells (i.e., the expression of stemness factors) using the teratocarcinoma cell line NT2. NT2 cells were chosen as the experimental system because unlike many other cancer cell lines, they retain the expression of stemness/reprogramming factors. Furthermore, they express both MYC and MYCN proteins. Our data showed that high-dose/short-term treatment of NT2 cells with Phenformin and Ibuprofen significantly down-regulated the expression of MYC/MYCN, OCT4, SOX2, and NANOG. Moreover, the anti-stemness effect of Ibuprofen in the low-dose/long-term treatment was also evident (0.15 mM at Day 7 of the drug-treatment or 0.15 mM and 0.25 mM at Day 4). Experiments are underway to address the similar effect of low-dose/long-term treatment of Metformin and Phenformin in NT2 cells. We are also investigating a change in global gene expression in the drugs-treated NT2 cells, with the emphasis on stem cell-related genes and pathways. Results of these studies will help us better understand how these common drugs (anti-inflammation and diabetes medications) can be used in anti-cancer therapeutics, and more importantly, in cancer prevention, which is the key to eradication of this disease.

Citation Format: Laura Grenlin, Tuan-Anh Tran, Sarah Lomahan, Naohiko Ikegaki, Xao X. Tang. Implications of the downregulation of stemness/reprogramming factor expression by ibuprofen and biguanides. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3858. doi:10.1158/1538-7445.AM2014-3858
Abstract 3859: MicoRNAs in regulation of colon cancer stem cells
Authors: Yu Yingjie , Pratima Nangia-Makker , Rajendra Sindhu , Levi Edi , Majumdar Adhip P. N.
Published: May 2015

Accumulating evidence supports the contention that epithelial cancers, including sporadic colorectal cancer (CRC), the incidence of which increases sharply after the age of 50 years, are diseases driven by self-renewing cancer stem/stem-like cells (CSCs). We have reported that CSC population in the colonic mucosa increases with advancing age. This was accompanied by a concomitant rise in microRNA-21 (miR-21) and reduction in miR-145 in the colonic mucosa. Interestingly, increased levels of miR-21 and reduction in miR-145 were noted in the colonic mucosa of patients with adenomatous polyps or CRC. These observations prompted us to speculate a role for miR-21 and miR-145 in regulating CSCs in the colonic mucosa. Indeed, we found that overexpression of miR-21 in colon cancer cells greatly increases CSCs and also induces tumor growth. An opposite phenomenon was noted in colon cancer cells where miR-145 was upregulated through stable transfection. In addition, studies utilizing SCID mice xenograft model of colon cancer showed that administration of either antagomir-21 (anti-sense miR-21) or excess miR-145 greatly inhibited the growth of tumors of colon cancer cells, when compared with the controls.

This reduction in growth was associated with decreased expression of CD44, B-catenin and Sox-2 and induction of CK-20. In vitro cell culture studies have further demonstrated miR-21 to regulate miR-145 and vice versa. Although the precise mechanism(s) by which miR-21 or miR-145 regulates CSCs in the colonic mucosa during the progression of CRC is unknown, our in vitro studies suggest that they induce differentiation of colon CSCs. The latter was evidenced by the increased expression of CK-20 in colon cancer cells following downregulation of miR-21 and upregulation of 145. In contrast, Sox-2 expression is decreased following overexpression of miR-145. In conclusion, our current observations suggest that miR-21 and miR-145, that are upregulated and downregulated, respectively, in the colonic mucosa during aging and CRC, play critical roles in regulating CSCs in the colon.

Citation Format: Yingjie Yu, Nangia-Makker Pratima, Sindhu Rajendra, Edi Levi, Adhip P. N. Majumdar. MicoRNAs in regulation of colon cancer stem cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3859. doi:10.1158/1538-7445.AM2014-3859
Abstract 3863: The stemness of gastric cancer stem cells is sustained by TGFβ produced from cancer-associated fibroblasts
Authors: Yashiro Masakazu , Hasegawa Tsuyoshi , Fukuoka Tatsunari , Kinoshita Haruhito , Morisaki Tamami , Kasashima Hiroaki , Masuda Go , Kubo Naoshi , Hirakawa Kosei
Published: May 2015

Purpose: Cancer-associated fibroblasts (CAFs) have recently been implicated in tumor growth and metastasis in gastric cancer. Cancer stem cells. (CSCs) have been proposed to have an important role in cancer progression. CSCs are known to display stem cell properties, “stemness”, the ability to sustain self-renewal. Because CAFs are abundant in the stroma of gastric cancer, CAFs are supposed to affect CSC characteristics of gastric cancer. However, the effects of CAFs on the characteristics of gastric cancer CSCs have not been clarified. The aim of this study was to clarify the effect of CAFs on CSCs characteristics in gastric tumor microenvironment.

Experimental Design: Four gastric cancer cell lines, OCUM-12, OCUM-2MD3, MKN-45, and MKN-74, were used. Side population (SP) cells were sorted by flow cytometry as CSC-rich cells from each parent cell line. CaF-37 was established from the tumoral gastric specimens as CAFs. Flow cytometric analysis of SP fraction, spheroid colony assay, and RT-PCR analysis of CSC markers, ABCG2, ALDH1, CD44, CD133, NANOG, and OCT3/4, were performed to identify CSCs properties. Effect of CAFs on the tumorigenicity by OCUM-12/SP cells was examined using nude mice.

Results: CaF-37 significantly increased the percentages of the SP fraction of scirrhous-type of gastric cancer cells, OCUM-12/SP and OCUM-2MD3/SP cells, but not that of non-scirrhous-type of gastric cancer cells, MKN-45/SP and MKN-74/SP cells. Taken together, CaF-37 significantly increased the number of spheroid colonies and the expression level of CSC markers of scirrhous-type of gastric cancer cells. These stimulating-activities by CaF-37 were significantly decreased by TGFβ inhibitors, but not FGFR and cMet inhibitor. Also, SP fractions of scirrhous-type of gastric cancer cells were significantly increased by TGFβ, but not by FGF2 and HGF. Tumorigenicity by subcutaneous co-inoculation of OCUM-12/SP cells with CaF-37 was significantly high in comparison with that by OCUM-12/SP cells alone. Phospho-Smad2 expression level was significantly increased by co-inoculation with CaF-37.

Conclusions: TGFβ from CAFs in the tumor microenvironment might sustain the stemness of CSCs of gastric cancer cells, especially in scirrhous-type of gastric cancer.

Citation Format: Masakazu Yashiro, Tsuyoshi Hasegawa, Tatsunari Fukuoka, Haruhito Kinoshita, Tamami Morisaki, Hiroaki Kasashima, Go Masuda, Naoshi Kubo, Kosei Hirakawa. The stemness of gastric cancer stem cells is sustained by TGFβ produced from cancer-associated fibroblasts. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3863. doi:10.1158/1538-7445.AM2014-3863
Abstract 3870: Effects of carbon ion beam, alone or in combination with cisplatin, on triple-negative breast cancer stem-like cells
Authors: Sai Sei , Karasawa Kumiko , Vares Guillaume Gueal , Shirai Toshiyuki
Published: May 2015

Aims

To investigate whether a carbon ion beam alone or in combination with cisplatin has beneficial effects compared to X-ray by targeting putative triple negative (TN) breast cancer stem cells (CSCs).

Materials and Methods

Human breast CSCs sorted from MDA MB231 cells were treated with carbon ion or X-ray irradiation alone or in combination with cisplatin, and then colony, spheroid and tumor formation assays, RT-PCR Array analysis as well as immunofluorescence γH2AX foci assay were performed.

Results

The colony, spheroid formation as well as tumorigenicity assays confirmed that CD44+/CD24- and CD44+/ESA+ cells exactly have CSC properties compared to CD44-/CD24- and CD44-/ESA- cells. CSCs were more highly enriched after X-ray or cisplatin compared to carbon ion beam. Carbon ion beam combined with cisplatin significantly suppressed colony and spheroid formation compared to that of X-ray combined with cisplatin as well as carbon ion alone. RT-PCR Array analysis showed that expression of DNA repair, apoptosis and autophagy related genes were differently altered between carbon ion beam and X-ray irradiation alone or in combination with cisplatin, and carbon ion beam combined with cisplatin more highly induced apoptosis and autophagy compared to X-ray combined with cisplatin. Immunofluorescence assay showed that not only the number but also the size of γH2AX foci in CSCs were lager 24 h after carbon ion beam combined with cisplatin compared to that of X-ray combined with cisplatin as well as carbon ion alone.

Conclusion

Carbon ion beam combined with cisplatin has superior potential to kill TN breast CSCs with unrepairable severe DNA damage and enhanced apoptosis as well as autophagy.

Citation Format: Sei Sai, Kumiko Karasawa, Guillaume Gueal Vares, Toshiyuki Shirai. Effects of carbon ion beam, alone or in combination with cisplatin, on triple-negative breast cancer stem-like cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3870. doi:10.1158/1538-7445.AM2014-3870
Abstract 3871: Tie-2 regulates stemness and metastasis of prostate cancer cells
Authors: Tang Kai Dun , Ling Ming-Tat
Published: May 2015

Introduction: Prostate cancer is the most commonly diagnosed male cancer in Western countries. Currently, the major treatment challenge for prostate cancer patients is the development of tumor metastasis. Ample evidence supports the idea that tumor metastasis originates from a rare population of cancer cells known as cancer stem cells (CSCs) [1]. Unfortunately, little is known about the identity of these cells, making it difficult to target prostate tumor metastasis. Here we reported the identification of a rare population of prostate cancer cells which express the Tie-2 protein, a tyrosine kinase receptor required for the bone marrow homing and colonization of hematopoietic stem cells. Notably, this Tie-2 positive population exists exclusively in highly metastatic prostate cancer cell lines.

Methods:To study the role of Tie-2 in prostate tumor metastasis, we have performed fluorescence-activated cell sorting (FACS) to isolate the Tie-2+ population from a prostate cancer cell line (PC-3). We then performed cDNA microarray analysis to characterize the gene expression profile of these cells. Furthermore, we have performed cell adhesion assay to examine the ability of the Tie-2+ cells in adhering to osteoblasts and endothelial cells. Besides that, we have also performed quiescent staining and drug sensitivity assay to examine whether Tie-2+ cells are more quiescent, and thus become resistant to chemotherapeutic drug. Finally, we have injected both Tie-2+ and Tie-2- PC-3 cells intracardiacly into the NOD-SCID mice to determine if Tie-2 expression promote prostate tumor metastasis under in vivo condition.

Results: Data from our study revealed that Tie-2+ cells express higher level of prostate CSC markers when compared to the Tie-2- population. Meanwhile, Tie-2+ cells are highly adhesive to both osteoblasts and endothelial cells, a characteristic necessary for tumor metastasis. We also found that Tie-2+ cells are more quiescent and resistant to the chemotherapeutic drug cabazitaxel, further support that these cells possess CSC-like characteristics. More importantly, we found that Tie-2+ cells, but not the Tie-2- cells population, developed metastatic tumor in vivo.

Conclusions: Our data suggested that Tie-2 plays an importantly role in the development of drug resistance and prostate tumor metastasis. Thus, Tie-2 might be a novel therapeutic target for treatment of advanced prostate cancer patients.

References:[1] J.E. Visvader, G.J. Lindeman, Cancer stem cells in solid tumours: accumulating evidence and unresolved questions, Nature reviews. Cancer, 8 (2008) 755-768.

Citation Format: Kai Dun Tang, Ming-Tat Ling. Tie-2 regulates stemness and metastasis of prostate cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3871. doi:10.1158/1538-7445.AM2014-3871
Abstract 3876: Can Nanog activity identify aggressive breast cancer stem cell populations
Authors: Sayers Thomas J. , Kluyver Rachel de , Kaddoura Marcella , Stauffer Jim , Tewary Poonam , Brooks Alan D.
Published: May 2015

Many tumors have been demonstrated to contain cancer stem cells (CSC). We hypothesized that expression of transcription factors that are required for induction and maintenance of pluripotency would be a possible alternative to cell surface phenotype for identification of CSC populations. Therefore we evaluated Nanog expression in 4T1.2 murine breast carcinoma cells, as a biomarker for CSC sub-populations. Introduction of a Nanog promoter reporter that drives destabilized GFP into 4T1.2 cells resulted in a stable expression of GFP in about 1-3% of the cells. On in vitro cell culture of FACS sorted GFP+ populations, up to 80% of the cells became GFP- over a period of 4 weeks, whereas GFP- populations remained 100% GFP-. In vitro CSC surrogate assays show that Nanog-GFP+ cells exhibited CSC characteristics, with enhanced colony formation in methylcellulose and increased survival in serum-free media. Nanog-GFP+ cells were also more efficient at generating experimental lung metastases following intravenous injection. In addition Nanog-GFP+ cells were also more resistant to the chemotherapeutic drugs paclitaxel and bortezomib. However targeting the extrinsic apoptosis pathway in vitro with a combination of bortezomib and agonist antibodies to the TRAIL death receptor DR5 was equally effective against both Nanog-GFP+ and Nanog-GFP- cell populations. To monitor Nanog expression in human breast cancer cells (SUM159), we used lentiviral vectors to introduce DNA constructs of the Nanog response element driving GFP and luciferase. About 0.5% of the SUM159 cells were GFP+ by FACS, and these cells also demonstrated some CSC characteristics such as enhanced colony formation in methylcellulose. Levels of GFP were increased in the SUM159 cells following cell culture under hypoxic conditions. These findings suggest that Nanog activity may be useful as surrogate marker of aggressive breast CSC subpopulations.

Funded by NCI Contract HHSN261200800001E

Citation Format: Thomas J. Sayers, Rachel de Kluyver, Marcella Kaddoura, Jim Stauffer, Poonam Tewary, Alan D. Brooks. Can Nanog activity identify aggressive breast cancer stem cell populations. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3876. doi:10.1158/1538-7445.AM2014-3876
Abstract 388: Epigenome-wide profiling identified significant differences in DNA methylation between African-American and European-American men with prostate cancer
Authors: Kwabi-Addo Bernard , Wang Songping , Devaney Joseph
Published: May 2015

Background: DNA methylation is a dynamic epigenetic mark that is essential for mammalian organismal development. For human prostate cancer (PCa), abundant evidence has accumulated to suggest that somatic epigenetic alterations may appear early during cancer development, as well as more commonly and consistently, than genetic changes. Several cancer-associated genes have been reported to show significant differences in DNA methylation pattern in prostate tissues from African-American (AA) and European-American men, but the genome-wide pattern and extent of these differences are largely unknown. The current study investigated genome-wide DNA methylation differences in AA and EA prostate tissue samples with the aim of assessing the variation of genome-wide patterns of DNA methylation.

Methods: To comprehensively examine the DNA methylation pattern in AA and EA samples, we used illumina 450 K methylation platform (Infunium Chip) to conduct genome-wide large scale analysis of DNA methylation changes in 7 normal and 3 PCa tissue samples from AA versus 8 normal and 3 PCa tissue samples from EA. This chip interrogates >485,000 CpG methylation sites per sample at single-nucleotide resolution and assigns each site an average beta value for quantitative methylation level.

Results: Pathway analysis of the genes with altered methylation patterns identified top canonical pathways for the involvement of cancer related network for genes involved in axonal guidance, antigen presentation, androgen signaling and protein ubiquitination pathways in prostate cancer tissues compared with normal prostate tissues obtained from AA men. On the other hand, the top canonical pathways identified in prostate cancer tissues compared with normal prostate tissues obtained from EA men are genes involved in epithelial-mesenchymal transition, p53 signaling, nucleotide sugars metabolism and germ cell-sertoli cell junction signaling pathways.

Conclusion: Using this quantitative sequencing-based approach, our work uncovers significant global DNA methylation alterations in AA versus EA prostate tissues and provides a mechanistic explanation for the disease disparity.

Citation Format: Bernard Kwabi-Addo, Songping Wang, Joseph Devaney. Epigenome-wide profiling identified significant differences in DNA methylation between African-American and European-American men with prostate cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 388. doi:10.1158/1538-7445.AM2014-388
Abstract 3889: Modulation of breast cancer stem cell marker CD44 by all-trans-retinoic acid (ATRA)
Authors: Kitadai Rui , Bednarczyk Robert , Tuli Neha , Hanly Elyse , Benrahoma Ghada , Suriano Robert , Mittelman Abraham , Tiwari Raj K.
Published: May 2015

Breast cancer stem cells (BCSCs), often characterized by cell surface marker expression as being CD44+/CD24- or CD44+/CD24low phenotypes, constitute a subpopulation of tumor cells that are highly tumorigenic. With respect to CD44, it has been reported that its expression is influenced by tumor microenvironment (TME) and that modulation of CD44 is detrimental to breast cancer stem cell self-renewal and differentiation. Interestingly, the factors that mitigate this effect are still unknown, which has led us to hypothesize that factors within the TME, comprising of differentiating agents, modulate the BCSC phenotype by regulating cell surface markers. MDA-MB 231 showed presence of BCSCs as CD44 positive cells were detected using immunofluorescence and Western blot analyses. The expression of CD44 on MDA-MB 231 was not responsive to the proinflammatory molecule IL-8 or estrogen. Both MDA-MB 231 and T47D were responsive to IL-8 and estrogen with respect to migration and invasion as examined in vitro using a Boyden Chamber assay suggesting that migration and invasion was independent of CD44 expression. Alternatively IL-8 responsiveness may be on a select clonal population of CD44- cells. The presence of differentiating agents within the TME has been explored. To examine the effect of differentiating agents, we used the classical all-trans-retinoic acid (ATRA) previously shown to promote tumor regression. CD44 expression was highly responsive to ATRA as it was down regulated following treatment. ATRA treatments also resulted in decreased migration and invasion of breast cancer cells. Our results led us to conclude that specific differentiating agents can affect breast cancer stem cell activity by directly down regulating key adhesion cell surface markers.

Citation Format: Rui Kitadai, Robert Bednarczyk, Neha Tuli, Elyse Hanly, Ghada Benrahoma, Robert Suriano, Abraham Mittelman, Raj K. Tiwari. Modulation of breast cancer stem cell marker CD44 by all-trans-retinoic acid (ATRA). [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3889. doi:10.1158/1538-7445.AM2014-3889
Abstract 3892: Inhibitory effects and regulatory mechanism of nestin in pancreatic cancer
Authors: Matsuda Yoko , Ishiwata Toshiyuki , Yoshimura Hisashi , Yamashita Satoshi , Ushijima Toshikazu , Takubo Kaiyo , Arai Tomio
Published: May 2015

Introduction: Nestin, a progenitor/stem cell marker, is expressed in human pancreatic cancer, and we have previously shown that its expression positively correlates with invasiveness and metastasis of the cancer (Cancer Biol Ther, 2011; Am J Pathol. in press). Here, we examined the inhibitory effects of nestin expression using nestin-targeted small interfering RNA (siRNA) in pancreatic cancer cells. We also investigated the regulatory mechanism of nestin expression in pancreatic cancer. Methods: siRNA targeting nestin was transfected to the pancreatic cancer cell lines PANC-1 and PK-45H, and we analyzed the effect on cell behaviors including proliferation, migration, invasion, sphere-forming ability, and sensitivity to gemcitabine. Next, to clarify the mechanisms of nestin expression, pancreatic cancer cells were injected into the spleen of NOD.Cg-Prkdcscid Il2rgtm1Sug/Jic (NOG) mice, and pancreatic cancer cells were also collected from metastatic foci of NOG mice. We compared the methylation status of the promoter and the 1st and 2nd introns of the nestin gene in metastatic and parental cancer cells using bisulfite sequencing and pyrosequencing.

Results: Nestin siRNA inhibited the proliferation, migration, invasion, and sphere-forming ability of the pancreatic cancer cells in vitro. Co-treatment of pancreatic cancer cells with gemcitabine and nestin siRNA decreased cell viability compared to administration of gemcitabine or nestin siRNA alone. Cells derived from the metastatic nodules of NOG mice showed higher expression of nestin compared with parental cells, and methylation of the nestin promoter and the 1st and 2nd introns was lower in the metastatic cells. Compared to control siRNA-treated mice, nestin siRNA significantly inhibited primary and metastatic tumor formation derived from human pancreatic cancer cells in an orthotopic implantation model using NOG mice.

In summary, decreasing nestin expression in pancreatic cancer cells using nestin siRNA suppressed cell proliferation, migration, invasion, and sphere formation in vitro and primary and metastatic tumor formation in vivo. Furthermore, nestin siRNA had synergistic effects with gemcitabine in inhibiting pancreatic cancer cell growth. These findings suggest that nestin plays a key role in pancreatic cancer cell metastasis and stemness, and that administration of nestin siRNA is a novel therapeutic strategy for pancreatic cancer.

Citation Format: Yoko Matsuda, Toshiyuki Ishiwata, Hisashi Yoshimura, Satoshi Yamashita, Toshikazu Ushijima, Kaiyo Takubo, Tomio Arai. Inhibitory effects and regulatory mechanism of nestin in pancreatic cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3892. doi:10.1158/1538-7445.AM2014-3892
Abstract 3899: ROCK inhibitor as a differentiation-inducing agent to target glioma stem cells
Authors: Auffinger Brenda , Tobias Alex L. , Han Yu , Lesniak Maciej S. , Ahmed Atique U.
Published: May 2015

Only 3% of patients diagnosed each year with Glioblastoma Multiforme (GBM) survive longer than 5 years, making GBM one of the most lethal forms of human cancer. It has been proposed that GBMs are driven by a rare subset of tumor cells referred to as cancer stem cells (CSCs), which have the ability to resist conventional radio- and chemotherapy and initiate disease recurrence. Thus, the development of novel and effective therapeutic strategies targeting CSCs is required and should significantly improve the dismal prognosis for patients with GBM. A possible viable strategy to effectively target glioma stem cells (GSCs) is the use of bone morphogenetic proteins (BMPs) as differentiation inducers. Our results suggest that Rho-associated protein kinase (ROCK) could work as a pharmacological negative regulator of BMPs. Our preliminary data indicate that: i) Fasudil, a ROCK1 inhibitor, induces BMP2 and BMP4 expression both in vitro and in vivo in different human-derived glioma xenograft models; ii) Fasudil-induced BMPs can increase the expression of differentiation markers and decrease stemness in these glioma models; iii) temozolomide, a chemotherapeutic agent, converts glioma cells to a GSC phenotype, while Fasudil blocks this interconversion; iv) co-administration of Fasudil with temozolomide inhibits the invasive characteristics of GBM in an orthotropic glioma xenograft model; and v) the analysis of BMP expression in human-derived GBM samples from the Repository of Molecular Brain Neoplasia Database (REMBRABDT) reveals that patients with higher BMP2 expression survived four times longer than those with low or intermediate BMP2 expression (p<0.001). Based on this, we propose that, by blocking ROCK signaling with the FDA approved pharmacological inhibitor Fasudil, one can modulate BMP expression in human-derived gliomas. Fasudil, therefore, may redirect cellular signals regulating GSCs' stemness, fate, and initiate their differentiation. Such an agent will allow us to develop a more effective anti-glioma therapy through its combination with other conventional anti-cancer drugs.

Citation Format: Brenda Auffinger, Alex L. Tobias, Yu Han, Maciej S. Lesniak, Atique U. Ahmed. ROCK inhibitor as a differentiation-inducing agent to target glioma stem cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3899. doi:10.1158/1538-7445.AM2014-3899
Abstract 391: Identification of a novel tumor-suppressor gene through methylome analysis in smoking-associated lung adenocarcinoma
Authors: Imoto Issei
Published: May 2015

Aberrant methylation of promoter DNA and transcriptional repression of specific tumor suppressor genes (TSGs) play an important role in carcinogenesis. In order to identify novel TSG candidates implicated in smoking-associated lung adenocarcinoma (AC), we performed genome-wide methylation profiling of six smoking-associated lung ACs with paired non-cancerous tissues using the single-CpG resolution Infinium array. After defining differentially methylated candidates including known targets in ACs, we analyzed gene expression using microarray/RT-PCR to identify genes potentially silenced by promoter methylation. Frequent hypermethylation and downregulation of candidates was further validated by pyrosequencing and RT-PCR/immunohistochemistry, respectively, in a larger set of ACs, and methylation dependency of silencing was determined by 5-aza-2-deoxycytidine treatment and promoter assay in AC cell lines. Through this approach, we identified one gene matching all of our selection criteria for a possible TSG (HSLAC1, Lab. name). Additionally, functional analyses supported a tumor-suppressive activity in vitro. Our results implicate HSLAC1 as a novel TSG for smoking-associated lung AC.

Citation Format: Issei Imoto. Identification of a novel tumor-suppressor gene through methylome analysis in smoking-associated lung adenocarcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 391. doi:10.1158/1538-7445.AM2014-391
Abstract 3910: Identification of lead organic compounds active against stem cell-like neuroblastoma cells by high throughput screening
Authors: Ikegaki Naohiko , Ratia Kiira , Hsiao Ruth , Limpar Mariko M. , Lomahan Sarah , Tang Xao X.
Published: May 2015

Cancer stem cells (CSCs) are plastic in nature, a characteristic that hampers cancer therapeutics. We previously reported the establishment of induced neuroblastoma (NB) stem cells (iCSC), which were stable stem cell-like NB cells in sphere culture. These cells recapitulated the in vivo histological phenotypes of Large-Cell NB (LCN), the most aggressive and deadly subset of NB, including vesicular nuclei and two to three prominent nucleoli (PNAS Vol. 110: 6097-102 2013). In addition to the LCN phenotype, high-level expression MYC or MYCN is the consistent feature of the iCSC xenografts grown in immune-compromised mice. The goal of this study is to identify lead organic compounds active against NB iCSCs by high throughput screening. By using MTS assays, the SKNAS iCSC and the parental monolayer cells were used to screen the Prestwick Chemical Library®, containing 1200 FDA-approved small molecules. Growth suppressive compounds were defined as they exhibited cell survival reduction of greater than 80% compared to DMSO control. There were unexplored compounds with significant antineoplastic activity among existing antibacterial, antifungal, antiprotozoal, and anthelmintic compounds. Among the compounds screened, we identified a dozen of small molecules that were preferentially growth suppressive to the SKNAS iCSC over the monolayer cells. The secondary MTS assay confirmed so far that Chlorhexidine (antiseptic), Benzethonium (antiseptic) and Digoxin (cardiac glycoside) were potential anti-NB iCSC agents: they were more effective in suppressing growth of the SKNAS iCSC than the monolayer cells. In addition, these compounds exhibited MYC destabilizing activity in the SKNAS iCSC. Notably, JQ1, the recently identified MYC destabilizer, was less effective than Chlorhexidine, Benzethonium and Digoxin in suppressing growth of SKNAS iCSC. The above three compounds also showed the MYCN destabilizing effect in another pair of MYCN-amplified SKNBE(2)C iCSCs and SKNBE(2)C monolayer cells. We are currently completing characterization of the small molecules identified with focus on their anti- iCSC growth and anti-MYC/MYCN effects. This study would reveal specific pathways that regulate biological activities of the CSCs. In addition, it is a promising approach that could lead to development of safe and effective anti-cancer therapeutics not only for NB but also other cancer types.

Citation Format: Naohiko Ikegaki, Kiira Ratia, Ruth Hsiao, Mariko M. Limpar, Sarah Lomahan, Xao X. Tang. Identification of lead organic compounds active against stem cell-like neuroblastoma cells by high throughput screening. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3910. doi:10.1158/1538-7445.AM2014-3910
Abstract 3917: Understanding the mechanisms driving pancreatic neuroendocrine tumorigenesis
Authors: Wasylishen Amanda R. , Lozano Guillermina
Published: May 2015

A recent exome sequencing study of pancreatic neuroendocrine tumors (PanNETs) provided valuable information about the genetic landscape of these cancers and for the first time implicated DAXX and ATRX loss in tumorigenesis. These interacting proteins are chaperones for the histone variant H3.3 and contribute to chromatin homeostasis and telomere maintenance. Mutually exclusive mutations in DAXX or ATRX occur in 43% of PanNETs. To understand how loss of these epigenetic regulators specifically contributes to tumorigenesis, we are developing genetically engineered mouse models of PanNETs. While a mouse with a conditional Daxx allele is publically available, subsequent sequencing and characterization revealed a mutant loxP site, which renders recombination extremely inefficient. We are, therefore, currently generating a new conditional Daxx allele, as well as using an existing conditional Atrx allele to specifically delete these genes in the endocrine pancreas. Cohorts of mice are being built and followed for developmental and tumor phenotypes. In addition, in vitro studies have also implicated a role for DAXX as a negative regulator of the p53 tumor suppressor pathway. Interestingly, p53 is rarely mutated in PanNETs. We are using a Daxx-null allele, generated using the existing conditional Daxx mouse, to investigate the relationship between DAXX and the p53 pathway in vivo. Combined, this work aims to generate and characterize mouse models which will lead to a better understanding of both the etiology of PanNETs and how loss of DAXX and ATRX contribute to tumorigenesis.

Note: This abstract was not presented at the meeting.

Citation Format: Amanda R. Wasylishen, Guillermina Lozano. Understanding the mechanisms driving pancreatic neuroendocrine tumorigenesis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3917. doi:10.1158/1538-7445.AM2014-3917
Abstract 3925: The PIK3CA
H1047R mutation can initiate tumorigenesis in the colon
Authors: Pasch Cheri , Maher Molly , Deming Dustin
Published: May 2015

Background: Our understanding of how mutations within the intestinal epithelium result in tumor development and progression is continuously evolving. We have previously described the FC PIK3ca* murine colon cancer model which expresses a constitutively activated phosphoinositide-3 kinase (PI3K) in the intestinal epithelium. The expression of this dominantly active form of PI3K resulted in invasive mucinous adenocarcinomas in the proximal colon through a non-canonical mechanism. We sought to determine if the hotspot PIK3CA H1047R mutation, commonly found in human colorectal cancers, can also induce intestinal tumorigenesis.

Methods: Female FVB mice carrying a conditional PIK3CA H1047R mutation (PIK3CAH1047R) were crossed to B6 males carrying atransgene in which the fatty acid binding protein promoter is fused to Cre recombinase (FC). (FVB x B6)F1 FC PIK3CAH1047R progeny express this constitutively active form of PI3K in epithelial cells of the distal small bowel and colon. 22 F1 FC PIK3CAH1047R mice were examined. Necropsy was performed at approximately 200 days of age or once moribund.

Results: 50% of FC PIK3CAH1047R mice develop invasive adenocarcinomas within the colon. These cancers are highly mucinous and often penetrate through the serosal layer of the colon. These lesions can be found throughout the colon, but are more common proximally. No tumors were identified within the small intestine. These cancers can have associated invasion into the regional lymphatics and metastases to the mesentery. Interestingly these cancers have a minimal luminal component compared to the amount of submucosal extension. Precursor lesions are able to be identified and are not polypoid. These cancers demonstrate activation of the AKT signaling pathway, but do not demonstrate nuclear localization of β-catenin.

Conclusion: This is the first report of the hotspot PIK3CA H1047R mutation leading to intestinal tumorigenesis through a non-canonical mechanism, resulting in flat mucinous invasive adenocarcinomas.

Citation Format: Cheri Pasch, Molly Maher, Dustin Deming. The PIK3CAH1047R mutation can initiate tumorigenesis in the colon. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3925. doi:10.1158/1538-7445.AM2014-3925
Abstract 3977: Molecular reprogramming in high-risk progressive neuroblastoma
Authors: Pandian Vijayabaskar , Khan Faizan H. , Ramraj Satish K. , Aravindan Sheeja , Natarajan Mohan , Herman Terence S. , Aravindan Natarajan
Published: May 2015

High risk disease in infants with neuroblastoma significantly contributes to the pediatric cancer death. We investigated the molecular reprogramming in neuroblastoma cells that drives favorable neuroblastoma to aggressive disease. For this, multi-site aggressive tumors from animals that received parental (SH-SY5Y) cell xenotransplantation were assayed and compared to the animals with subtle non-metastatic xenografts. Establishment of highly malignant neuroblastoma cell line (NBP4) and its comparative (with SH-SY5Y) cytogenetic/phenotypic physiognomies were reported elsewhere. Ex vivo whole genome gene expression identified a total of 33 genes upregulated and another 33 genes downregulated (>40 fold change) in NBP4 cells as opposed to parental SH-SY5Y cells. QPCR analysis revealed a conforming upregulation of SEMA3D, MEGF10, GRB10 and suppression of RD3 in multi-site aggressive tumors. Tissue microarray (72 core) constructed with aggressive multiple tumors and non-progressive controls coupled with immunohistochemistry showed robust levels of SEMA3D, GRB10, MEGF10 and complete loss of KCNQ2, KRTAP1-1 and RD3. Consistently, translational modifications of SEMA3D, GRB10, MEGF10, KCNQ2 and RD3 in multi-site aggressive tumors were validated with immunoblotting. More importantly, selective knockdown of KCNQ2 or KRTAP1-1 in parental SH-SY5Y cells robustly modified the cellular phenotype and significantly increased the tumorosphere formation. Conversely, discrete muting of GRB10, KIAA2022, MEGF10 or SEMA3D ex vivo (in aggressive NBP4 cells) endorsed the cellular differentiation, adherence and resulted in complete regulation of tumorosphere formation. Together, these data suggests that the milieu based molecular reprograming of the neuroblastoma cells drives the development of high-risk disease and further implies that GRB10, KIAA2022, MEGF10, SEMA3D, RD3 and KCNQ2 may concert and orchestrate the disease progression. Signal flow-through and molecular driver(s) that orchestrate favorable to high-risk disease are yet to be understood and are currently in progress in our laboratory.

Citation Format: Vijayabaskar Pandian, Faizan H. Khan, Satish K. Ramraj, Sheeja Aravindan, Mohan Natarajan, Terence S. Herman, Natarajan Aravindan. Molecular reprogramming in high-risk progressive neuroblastoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3977. doi:10.1158/1538-7445.AM2014-3977
Abstract 398: Epigenetic changes in testis specific Y-like 5 gene in human prostate carcinoma: Gene expression analysis and its potential as a biomarker
Authors: Kumar Senthil R. , Bryan Jeffrey , Esebua Magda
Published: May 2015

INTRODUCTION

Testis specific Y-like protein (TSPYL-5) is a member of TSPY-L family of gene, whose functions are currently unknown. TSPYL-5 was reported to possess tumor suppressor function and a target for epigenetic silencing in human glioblastoma and colon carcinoma. The exact nature of TSPYL-5 gene expression and its epigenetic modification is not known in prostate carcinoma. We hypothesize that this gene could be expressed in prostate carcinoma cells and tissues and its methylation status could function as an epigenetic biomarker for aggressive prostate cancer phenotype.

MATERIALS AND METHODS

Human prostate carcinoma cells (PC3, DU145, LnCAP) was grown in DMEM-F12 media with 10% FBS at 37oC. Normal prostate epithelial cells (RWPE1-) were maintained in keratinocyte serum free media. Total RNA was extracted from the cells and converted to cDNA which served as a template for PCR amplification in the presence of TSPYL-5 gene primers. The amplified PCR products were analyzed by 2% agarose gel electrophoresis. Methylation-specific PCR (MSP) was performed with primers designed for the CpG island in the gene promoter to detect the methylation changes in the carcinoma cells.

RESULTS

Quantitative RT-PCR analysis of TSPYL-5 gene expression in different human prostate carcinoma cells lines identified 2-3 fold higher expression (P ≤ 0.005) in lines with metastatic propensity (PC3) compared to less aggressive ones. MSP analysis indicated differential promoter methylation of TSPYL-5 gene in carcinoma and normal prostate epithelial cells (RWPE1). Consistent methylation in LnCAP and DU145 cells was observed in our experiments and was associated with reduced expression of the gene.

CONCLUSIONS

Expression of TSPYL-5 is higher in human prostate carcinoma cells derived from more metastatic phenotype. This expression could be influenced by DNA methylation. It is not clear whether TSPYL-5 expression in prostate carcinoma cells could play a part in tumor suppressor function. Alternately, the presence of this gene may play alternate roles including treatment resistance and cell growth. Further, DNA methylation of the CpG island of TSPYL-5 could serve as a robust biomarker of aggressive phenotype in human prostate carcinoma.

Citation Format: Senthil R. Kumar, Jeffrey Bryan, Magda Esebua. Epigenetic changes in testis specific Y-like 5 gene in human prostate carcinoma: Gene expression analysis and its potential as a biomarker. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 398. doi:10.1158/1538-7445.AM2014-398