bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 3283: Conditional Low-molecular-weight (LMW) cyclin E-induced mammary tumorigenesis
Authors: Akli Said , Van Pelt Carolyn S. , Wenbin Liu , Keyomarsi Khandan
Published: August 2012

Cyclin E overexpression and generation of hyperactive LMW cyclin E is oncogenic and has been implicated in a variety of human cancers. We have developed a doxycycline-inducible LMW cyclin E transgenic mouse model to determine the dependence of established mammary adenocarcinomas on continued expression of LMW cyclin E. Mice harboring this TetO-LMW-E transgene, referred to as TLMW, were mated to MMTV-rtTA transgenic mice (MTB) to yield bitransgenic MTB/TLMW mice. Morphological examination of hematoxylin and eosin-stained sections showed that the mammary glands of induced MTB/TLMW mice following four days of doxycycline induction were hyperplastic compared with uninduced MTB/ TLMW animals. Consistent with this, immunohistochemistry using antibodies specific for BrdU revealed a 2-fold increase in BrdU incorporation in the mammary epithelium of induced bitransgenic females compared with uninduced mice (17.1+/−5.3% versus 30.9+/−4.9%, P=0.03). This data indicate that LMW cyclin E overexpression in the mammary epithelium increased proliferation. To determine whether sustained induction of transgene expression would result in mammary tumorigenesis in MTB/TLMW mice, a cohort of 27 bitransgenic females was treated with doxycycline beginning at 6 weeks of age. 41% of these mice developed tumors within 2 years of starting doxycycline with kinetics (T1/2= 20 months) resembling that described for MMTV-LMW cyclin E mice. Similarly, the predominant type of tumor observed for the inducible LMW forms was glandular adenocarcinomas with adenosquamous differentiation. Lung metastases are known to arise in a fraction of MMTV-LMW cyclin E transgenic mice harboring primary mammary tumors. Accordingly, we sacrificed a subset of mammary tumor bearing MTB/TLMW mice and examined organs at necropsy for evidence of metastatic spread. 4 of 7 mice examined had lung metastasizes (57%). Control mice without dox never developed mammary tumors. To shed further light on the molecular mechanisms of mammary tumorigenesis caused by LMW-E expression, we conducted DNA microarray analyses. Using the R package limma, 641 probes out of 30,854 probes were identified as differentially regulated by at least 2-fold by LMW-E in the tumors when compared to the contralateral mammary glands. Gene Ontology analysis identified genes on this list that were involved in a variety of processes, including cell cycle control of chromosome replication, and DNA methylation and transcriptional repression signaling. Based on histology and gene expression profile of the resultant mammary tumors, we propose that the MTB/TLMW is a murine model of breast cancer resembling the human invasive basal/triple negative breast cancer.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3283. doi:1538-7445.AM2012-3283
Abstract 3286: Interleukin-17 receptor c (IL-17RC) knockout mice developed fewer and smaller prostate tumors compared to the wild-type mice in Pten-deficient context
Authors: You Zongbing , Zhang Qiuyang , Liu Sen , Ge Dongxia , Zhang Qingsong , Xue Yun
Published: August 2012

Introduction: Interleukinα17 (ILα17) is a key cytokine in inflammatory and immune diseases. ILα17 may promote or inhibit tumor growth. Several studies have linked ILα17 with prostate cancer. ILα17's role in prostate cancer is unknown. The objective of this study is to investigate ILα17's role in formation and growth of prostate adenocarcinoma. Methods: ILα17 receptor C (ILα17RC) knockout mice were crossed with Pten conditional knockout mice (using ProbasinαCre). Ptenα/β male mice with intact ILα17 signaling (named RC+ mice, including ILα17RC+/+ and ILα17RC+/−) were compared with Ptenα/β male mice without ILα17 signaling (ILα17RCα/α, named RCβ mice). Results: There was no difference between the weight of genitourinary bloc of RC+ mice and that of RCβ mice at 4, 6 and 9 weeks. At 12 and 30 weeks, the GUαbloc weight of RC+ mice was 14% and 47% heavier than that of RCβ mice, respectively (P = 0.005, N = 54, and P = 0.004, N = 32, respectively). We found similar prostatic epithelial hyperplasia at 4 weeks and prostatic intraepithelial neoplasia (PIN) at 6 weeks in both RC+ and RCβ mice, suggesting that ILα17RC knockout did not affect PIN formation. At 9 weeks, invasive cancer was formed in 87% of RC+ prostate lobes, but only in 25% of RCβ prostate lobes (P < 0.001). At 30 weeks, 100% of RC+ prostate lobes formed invasive cancer, whereas only 70% of RCβ prostate lobes showed invasive cancer (P < 0.005). This suggests that ILα17RC knockout decreased progression of PIN to invasive cancer. The RCβ PINs were surrounded with a thick layer of fibromuscular stroma, whereas the stroma layer was significantly thinner in RC+ prostate glands, suggesting that failure of PIN to invade stroma was a critical deficit in RCβ mice. We found that MMP7 expression was significantly decreased in RCβ prostates compared to RC+ prostates, suggesting that lack of MMP7 expression is likely responsible for the observed phenotype. Furthermore, we found that ILα17 treatment induced MMP7 mRNA and protein expression in exαvivo cultured RC+ prostate tissues but not in RCβ prostate tissues, suggesting that MMP7 is a downstream target gene of ILα17 signaling. Conclusions: ILα17 promotes prostate adenocarcinoma formation and growth through induction of MMP7 expression in mouse prostate. Funding provided by NIH/NCRR COBRE Grant (2P20RR020152α06), DoD (W81XWHα05α1α0567 and W81XWHα10α1α0937), Tulane Cancer Center, Louisiana Cancer Research Consortium, and Tulane Framework for Global Health Seed Grant.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3286. doi:1538-7445.AM2012-3286
Abstract 3295: Systematic in vivo analysis of PI3K pathway aberrations in a mouse model for invasive lobular carcinoma
Authors: van Miltenburg Martine H. , Klarenbeek Sjoerd , Braumuller Tanya , Henneman Linda , Ali Rahmen Bin , Huijbers Ivo , Krimpenfort Paul , Berns Anton , Jonkers Jos
Published: August 2012

Invasive lobular carcinoma (ILC) is a form of breast cancer that develops in the milk-producing glands (lobules) of the breast. ILC is an aggressive form of breast cancer due to its ability to spread to other parts of the body, both by bloodstream as well as lymphatic system. One of the key features found in ILC is loss of E-cadherin, encoding CDH1. Mammary-gland specific deletion of E-cadherin does not lead to tumour formation indicating that other factors are involved in the induction of ILC. Indeed, combined tissue-specific inactivation of E-cadherin and p53 induces development of invasive and metastatic mammary tumours resembling human ILC. However, a limitation of the current conditional mouse models is that human ILCs rarely contain p53 mutations. In contrast, PI3K pathway mutations are frequently found in human ILCs. Currently we are developing novel mouse ILC models to systematically examine the contribution of PI3K pathway components such as PIK3CA, AKT and PTEN to ILC development. To reduce time spent on generating a genetically engineered mouse (GEM) tumour cohort expressing three or more transgenes we set-up embryonic stem (ES) cell derivation and targeting of ES cells from validated GEM models, the so-called GEMM-ESC strategy. WapCre-Cdh1F/F ES cells targeted with PIK3CA or AKT mutants are now being used to generate the elaborate PI3K pathway tumour cohort. These advanced models will allow us to study the role of the PI3K pathway in the pathogenesis of ILC. Moreover, we can use these advanced mouse models which recapitulate key hallmarks of human ILC to perform refined and realistic preclinical prevention and intervention studies for ILC.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3295. doi:1538-7445.AM2012-3295
Abstract 3296: SRC-3 is a critical mediator in the development of castration-resistant prostate cancer
Authors: Tien Jean C. , Liu Zhaoliang , Gao Li , Wang Fen , Xu Jianming
Published: August 2012

Androgen ablation therapy is the standard treatment for advanced prostate cancer. While tumors initially respond to androgen deprivation, they almost always recur with an androgen-independent phenotype termed castration-resistant prostate cancer (CRPC). Castration-resistant tumors are incurable with current therapy regimens and account for most prostate cancer mortality. Better understanding of their biology is therefore critical for devising novel treatment options. Steroid Receptor Coactivator 3 (SRC-3) is a nuclear receptor coactivator that promotes growth of endocrine tissues. It enhances proliferation of prostate cancer cell lines and is highly expressed in advanced human prostate tumors. Despite this, the role of SRC-3 in CRPC is not well studied. PTEN is a tumor suppressor gene mutated in most human prostate cancers. Mice harboring PTEN deletion in prostatic epithelial cells develop cancer that arises from progenitors in the luminal epithelial and basal compartments. Tumors histologically mimic advanced human disease and are castration-resistant. We hypothesized that, in prostate tumors caused by PTEN deletion, SRC-3 is a critical mediator for accelerating the development of CRPC. To test this hypothesis, we generated mice in which floxed PTEN and SRC-3 genes were concomitantly deleted in prostate epithelial cells via a Cre recombinase driven by the Probasin promoter. We first compared tumor mass, histology and biomarkers in these PTENf/f/SRC-3f/f/ARR2PBiCre (pten3cko) mice vs. PTENf/f/ARR2PBiCre (ptencko) mice. We found that while tumor growth in pten3cko mice was slightly inhibited at 9 wks of age, tumor histology and cellular composition were not markedly different. We then castrated mice of both genotypes at 9-wks-old and harvested tumors at 12wks. Interestingly, castrated ptencko mice had significantly worsened tumor aggressiveness–as indicated by increased proliferation index, reduced differentiation (increased p63; decreased K5 and K8) and increased reactive stroma (double staining of SMA and vimentin)–versus their non-castrated counterparts. Amazingly, in addition to significantly reducing tumor size, SRC-3 ablation reversed all changes comprising the aggressive phenotype seen in the post-castration tumors. Further investigation showed SRC-3 deletion decreased mTOR signaling. Together, these data show that castration induces a worse tumor phenotype in the setting of PTEN deletion and that SRC-3 promotes this process by inducing epithelial cell proliferation and enhancing the growth-promoting effect of these cells on the stroma. Therefore, SRC-3 may serve as a potential target for controlling CRPC development.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3296. doi:1538-7445.AM2012-3296
Abstract 3298: Arsenic induces human bronchial epithelial cell transformation featured with tumor-initiating cells
Authors: Chang Qingshan , Chen Fei , Lu Yongju
Published: August 2012

Arsenic is a well-known human carcinogen. Chronic exposure to arsenic is associated with cancers in various tissues, including skin, urinary bladder, lung, liver, kidney and prostate. However, the mechanism underlying arsenic-induced carcinogenesis remains elusive. Here, we demonstrate that long-term (six month) exposure to low levels of sodium arsenite (0.25 µ M) in human bronchial epithelial cell line (BEAS-2B) results in altered morphology, increased cell proliferation and anchorage-independent colony formation in soft agar, indicative of malignant transformation of these cells. Moreover, tumorigenesis analysis revealed that s.c. inoculation of the arsenic-transformed cells can induce tumors in nude mice. The tumors can be observed after 2-3 weeks even only 500 arsenic-transformed cells were injected, suggesting that these transformed cells induced by long-term and low-dose arsenic treatment are highly tumorigenic. Additional studies indicated that some of these arsenic-transformed cells exhibit characteristics of tunor-initiating cells (TICs) or cancer stem cells (CSCs), such as overexpression of Oct3/4, Nanog, Sox2, Klf4, and C-myc. Tumor sphere formation assay, a general procedure in defining TICs or CSCs, suggested that about 5% of these arsenic-transformed cells can form tumor spheres in non-adherent plates. The tumor sphere forming cells were enriched through serial passage of the cells collected from tumor spheres. Flow cytometry sorting showed that 90% of these sphere cells are CD49f positive, whereas CD133, CD44, EpCAM, and c-kit were not detected. Overexpression of CD49f was confirmed by immunoblotting, which indicated an increased CD49f level in the arsenic-transformed cells relative to the non-transformed BEAS-2B cells. Finally, the ability of sphere formation and colony formation in soft agar was compared between the cells showed the strongest and the weakest CD49f expression and suggested that the cells with the strongest CD49f expression are more capable of forming tumor spheres and colonies. In conclusion, our data suggested that the transformed cells induced by arsenic have features of TICs or CSCs.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3298. doi:1538-7445.AM2012-3298
Abstract 3299: Hematopoietic chimerism in liver transplantation patients and hematopoietic stem/progenitor cells in adult human liver
Authors: Wang Xiao Qi , Lo Chung Mau , Chen Lin , Cheung Cindy KY , Fan Sheung Tat
Published: August 2012

Liver transplantation (LT) is a cure for many liver diseases. Blood chimerism of donor origin can develop after LT, which raises the possibility of existence of hematopoietic stem/progenitor cells (HSPCs) in liver. We characterized the blood chimerism in a large cohort of 226 LT patients and analyzed putative HSPCs in adult human livers. The overall incidence of chimerism was 6.19%, of which 10.3% was among short-term (one day to six months) and 3.6% was among long-term (six months to eight years) LT patients. Hematopoietic LinαCD34+CD38αCD90+ population has been demonstrated to generate long-term multipotent grafts in transplantations. In human adult livers, we detected LinαCD34+CD38αCD90+ population accounting for 0.056% ± 0.05% of the total single liver cells and for 0.06% ± 0.05% of CD45+ liver cells. Both LinαCD34+ and LinαCD45+ liver cells from extensive perfused human liver graft were capable of forming hematopoietic myeloid-lineage and erythroid-lineage methylcellulose colonies. More importantly, LinαCD45+ or CD45+ liver cells could be engrafted into hematopoietic cells in an immunodeficient mouse model. These results are the first evidence of presence of the putative HSPC population in adult human liver, where liver is a good ectopic niche. The discovery of existence of HSPCs in adult liver will have new implications for understanding of extra-marrow hematopoiesis, liver regeneration, mechanisms of tolerance in organ transplantation and de novo cancer recurrence in LT patients. Conclusion: Human adult liver contains small population of HSPCs. In LT patients, there are two types of chimerism: transient chimerism resulting from mature leucocytes and long-term chimerism deriving from putative HSPCs in the liver graft.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3299. doi:1538-7445.AM2012-3299
Abstract 3302: Roles of cortical and subventricular GFAP+ astrocytes in initiation of astrocytomas
Authors: Schmid Ralf S. , Bash Ryan E. , Werneke Andrea M. , Miller C. Ryan
Published: August 2012

Astrocytomas, including glioblastomas (GBM), are characterized by cellular, molecular, and morphological heterogeneity, which raises the question from which cells astrocytomas originate. Research with many different types of cancer has established that tumor initiating cells share molecular characteristics and gene expression signatures with stem cells; however, the origin of glioma initiating cells and their relationship to adult neural stem cells remains unclear. Using conditional, inducible genetically-engineered mouse models (GEMM) with inactivated RB (GFAP-driven transgenic expression of N-terminal SV40 large T mutant (T121), T) and/or PTEN (P) and/or constitutively activated KRAS (R, KRAS(G12D)), we show that tumorigenesis was initiated in both parenchymal GFAP+ astrocytes as well as GFAP+ NSC/progenitor cells in the subventricular zone (SVZ) within two weeks of induction in adult mice. The induced GFAP+ cells proliferated (measured by EDU incorporation) and expressed multiple neural stem cell (NSC) markers such as Sox2, nestin and CD133, which persisted in developing astrocytomas over several months. Primary cortical astrocytes from TRP-/- mice cultured in vitro under NSC conditions displayed self-renewal and multi-lineage differentiation potential and expressed multiple NSC markers. When stereotactically injected in the diencephalon of syngeneic recipient mice, astrocytomas formed within 3 weeks, which histopathologically and genetically resembled human glioblastoma and proved to be fatal with a median survival of 25 days. To further evaluate the relationship between adult neural stem/progenitor cells in the SVZ and astrocytoma initiating cells, we crossed TRP GEMM with knock-in Sox2-GFP reporter mice to purify TRP neural stem cells from the SVZ. These cells were then stereotactically injected into the diencephalon of syngeneic recipient mice. Allograft cells, identified by GFP expression, were found proliferating around the injection site several weeks after injection, suggestive of developing astrocytomas. Some cells also migrated along white matter tracks and infiltrated the stem cell niche in the subventricular zone. These results suggest that GFAP+ NSC within the SVZ, and potentially GFAP+ astrocytes elsewhere in the neuraxis, may serve as the cell-of-origin for diffuse astrocytomas in the adult murine brain.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3302. doi:1538-7445.AM2012-3302
Abstract 3304: Hyaluronan mediates interactions between CD44+ MSCs and CD34+ fibrocytes in Desmoid Tumors.
Authors: Rizvi Hira , Carothers Adelaide M. , Bertagnolli Monica M. , Cho Nancy L.
Published: August 2012

We reported that 16/16 sporadic and familial desmoid tumors (DTs) contained CD44+CD73+CD90+Bmi1+ mesenchymal stromal cells (MSCs) and CD34+CD105+ALK1+ monocyte-derived fibrocytes. Hematopoietic stem cells (HSCs) and MSCs reside in the same bone marrow niche, and MSCs regulate HSC progenitor release into the circulation. Numerous signaling pathways maintain chemotactic and physical crosstalk between these types of stem cells (c-Kit/SCF, CXCR4/SDF-1, Notch/Jagged1, etc). HSC and MSC progenitors are both recruited to sites of tissue injury to aid in wound healing. We were interested to learn what crosstalk engages these multipotent cells in DTs. Hyaluronan (HA) is a long charged chain of repeating glucuronate and N-acteylglucosamine disaccharides that is synthesized by HA synthases (HAS 1, 2, 3) and degraded by hyaluronidases (HYAL1, 2). HA binds to receptors CD44, TLR2/4 and Hyaluronan binding protein (HABP). Immunofluorescent histochemistry was performed on our tumor collection to determine the expression of both metabolic and catabolic HA enzymes. All of our DTs contained cells expressing HA, HYAL2, HAS2, and HABP2. HABP2 attenuates hyaluronan-receptor interactions. Double staining showed that all MSCs expressed HA and HAS2, confirming the functional significance of the CD44+ receptor in these cells. The majority of MSCs co-expressed HABP2. CD34+ fibrocytes but not CD44+ MSCs co-expressed HYAL2. CD44 expression by MSCs is stimulated by PDGF-BB, and in DTs, CD44+ MSCs co-expressed PDGFRα. These results suggest that paracrine regulation of HA signaling in these tumors may be another way in which MSCs and fibrocytes crosstalk.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3304. doi:1538-7445.AM2012-3304
Abstract 3308: Axl is required for EMT-induced stem cell traits and metastasis in breast cancer
Authors: Tiron Crina , Stefansson Ingunn , Haugen Hallvard , Sandal Tone , McCormack Emmet , Gjertsen Bjørn Tore , Micklem David , Ivaska Johanna , Akslen Lars , Lorens James
Published: August 2012

Epithelial-to-mesenchymal transition (EMT) endows carcinoma cells with migratory, survival and stem cell-like attributes that facilitate metastasis. We have shown that expression of the receptor tyrosine kinase Axl in primary breast cancer is a strong independent predictor of poor overall survival and is further enhanced in matched patient metastases. Axl is upregulated by EMT-induction in mammary epithelial cells and establishes an autocrine signaling loop with its ligand, Gas6. Axl signaling is required to maintain the mesenchymal and stem cell-like phenotype induced by EMT-transcription factors. Axl-inhibition downregulated mesenchymal and stem cell marker expression, blocked invasiveness, inhibited mammosphere formation and reversed drug resistance. Axl knockdown completely prevented the spread of metastatic breast carcinoma cells from the mammary gland and increased overall survival in two different orthotopic breast cancer models. Axl was also required for breast cancer cells to extravasate into the lung following intravenous injection. These results suggest that Axl signaling is necessary for metastasis, stem cell and therapy resistance traits in breast cancer. Hence, Axl-inhibition represents a novel therapeutic opportunity to target stem-like and drug resistant breast cancer cells.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3308. doi:1538-7445.AM2012-3308
Abstract 3309: Blocking the Notch pathway inhibits the epithelial-mesenchymal transition (EMT) status in lung cancer and alters chemoresistance
Authors: Hassan Khaled A. , Wang Luo , O'Dowd Paige , Kim Gwangil , Korkaya Hasan , Davis April , Liu Suling , Kalemkerian Gregory P. , Wicha Max
Published: August 2012

Background: The Epithelial-Mesenchymal Transition (EMT) is a key developmental program that is often activated during cancer invasion and metastasis. EMT cells have stem cells properties and possess the characteristics of cell motility, invasiveness and chemotherapy resistance. EMT expression profile correlates with poor outcome in multiple tumors and there is evidence suggesting an integral role of Notch pathway in mediating EMT. In this study, we investigated Notch activity and EMT status and the correlation with chemoressitance in non-small cell lung cancer (NSCLC). Results: Transduction of multiple NSCLC cell lines, NCI-H1299, NCI-H460, NCI-H358, NCI-H441 with a Notch GFP-reporter construct identified a subset of cells with high Notch activity (GFP-pos). We compared the tumor invasivness capacity of GFP-pos and GFP-neg cells utilizing a transwell invasion assay. Cells with high Notch activity, as evidenced by GFP expression, had multiple fold higher invasion capability compared to GFP-neg cells (p=0.0001). To evaluate whether Notch activity correlates with resistance to chemotherapy, we treated GFP-pos and GFP-neg cells with docetaxel or cisplatin and assessed Annexin V expression using flow cytometry. GFP-pos cells had five fold less apoptotic cells as compared to GFP-neg cells indicating resistance of GFP-pos cells to chemotherapy. When GFP-pos cells were exposed to a Notch signaling inhibitor inhibitor, gamma secretase inhibitor (GSI), these cells were rendered sensitive to cisplatin and docetaxel. RNA microarray analysis and RT-PCR analysis revealed an increased expression of EMT related genes (Vimentin, N-Cadherin, Snail-1,….) in GFP-pos cells as compared to GFP-neg cells. Furthermore, NOD/SCID mice with subcutaneous lung tumor xenografts treated with GSI showed decreased expression of downstream effectors of Notch as well as vimentin. This combination of GSI and docetaxel reduced tumor grwoth in mouse xenographs compared to GSI and docetaxel as single agents. Conclusion: These studies suggest that the Notch pathway is an important regulator of EMT status and that its inhibition may alter EMT chemo-resistance properties. The addition of a Notch inhibitor may enhance the efficacy of chemotherapy in the treatment of NSCLC.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3309. doi:1538-7445.AM2012-3309
Abstract 3310: microRNAs regulate the transition between EMT and MET breast cancer stem cell states
Authors: Liu Suling , Shang Li , Bai Shoumin , Martin-Trevino Rachel , McDermott Sean , Ginestier Christophe , Ibarra Ingrid , Baker Trenton , Malik Fayaz , Ke Jia , Patel Shivani H. , Luther Tahra , Korkaya Hasan , Zhang Kevin J. , Clouthier Shawn G. , Hannon Gregory J. , Wicha Max S.
Published: August 2012

Recent studies have suggested similarities between cancer stem cells and the epithelial mesenchymal transition (EMT) state. In contrast, other studies suggest that these “states” are mutually exclusive. Our studies suggest that these divergent views may be explained by the existence of multiple stem cell states, which are regulated by microRNAs. Utilizing primary breast tissue and established cell lines, we demonstrate that both normal and malignant breast stem cells exist in distinct, inter-convertible states. The EMT-like state is characterized by expression of vimentin and N-cadherin, slug, snail and twist transcription factors. EMT-like CSCs have a mesenchymal morphology, are largely quiescent, invasive and characterized by expression of the CSC markers CD44+CD24− and are EpCAM−CD49f+. In contrast, the MET (mesenchymal epithelial transition) state of CSCs is characterized by an epithelial morphology and expression of E-cadherin and EpCAM. MET-like CSCs undergo self-renewal and express the CSC marker Aldehyde dehydrogenase (ALDH) and are EpCAM+CD49F+. A subpopulation of cells expressing both CD44+CD24− and ALDH may represent cells in transition between these states. This transition is regulated by signals in the microenvironment which in turn modulate microRNA networks. Expression induction of mir100 in MCF10A cells and several cancer cell lines resulted in a decrease of ALDH-positive CSC population with a concomitant increase in the CD24−CD44+ population accompanied by induction of EMT. We demonstrated mir100 effects are mediated by targeting BMPR2, SMARCA5 and SMARCD1, all of which may contribute to induction of EMT. Moreover, we show that mir100 overexpression induces cellular quiescence as shown by Ki67 and Brdu staining. Induction of mir100 expression immediately upon orthotopic implantation or after the tumors are established significantly reduced the subsequent tumor growth in NOD/SCID mice. In contrast of overexpression of mir100 stimulates cell invasion in vitro matrigel assay. The existence of multiple cancer stem cell states has important implications for understanding stem cell plasticity as well as tumor growth and metastasis. In addition, the existence of these states has implications for the development of CSC-targeting therapeutics.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3310. doi:1538-7445.AM2012-3310
Abstract 3312: Targeting Notch signaling to abrogate the increase in breast cancer stem cells induced by hypoxia
Authors: Conley Sarah J. , Gheordunescu Elizabeth , Theisen Rebecca , Wicha Max
Published: August 2012

There is increasing evidence from our lab and others supporting the “cancer stem cell” hypothesis in breast cancer, which deems that tumors are driven a small population of cells exhibiting stem cell properties. These cancer stem/progenitor cells may also be responsible for mediating tumor metastasis and resistance to cancer treatments. Recently, several new cancer therapies targeting tumor angiogenesis have been developed and validated in clinical trials. However, clinical practice has revealed that these therapies ultimately result in drug resistance and do not prolong patient survival by more than a few months. It has been reported that these angiogenesis inhibitors promote tumor invasiveness and metastasis in rodent breast cancer models. We have used in vitro and in vivo assays to study the role of hypoxia in regulating mammary cancer stem cells. We have shown that breast tumor stem cell population increases in response to hypoxia in vitro and in tumors from anti-angiogenic treated mice. We hypothesize that this leads to increased tumor aggressiveness and may explain the resistance to these drugs seen in patients. In vivo, both sunitinib and bevacizumab treatment increased intratumoral hypoxia as well as the percentage of cells expressing ALDH as assessed by the Aldefluor assay in SUM-159 and MDA-MB-231 breast cancer xenografts in NOD/SCID mice. In addition, tumor cells obtained from sunitinib treated mice exhibit enhanced growth of secondary tumors in NOD/SCID mice, further supporting an increase in the tumor stem cell population. We also demonstrate that hypoxia (1-2% oxygen) enriches for stem cells by at least 2 fold in several breast cancer cell lines grown in vitro. Utilizing siRNA, we identified that HIF1a is required for the increased cancer stem cell population following hypoxia treatment. We next tested whether a gamma secretase inhibitor (GSI) can block hypoxia's ability to increase the cancer stem cell population since HIF1a is reported to activate the Notch signaling pathway, and this pathway is known to regulate stem cell self renewal. Treatment of cells with the GSI completely abrogated the increase in ALDH+ cells under hypoxic conditions. We are now testing the ability of GSIs to block the increase in CSCs seen in mice treated with anti-angiogenic drugs. This study may reveal that therapies targeting tumor angiogenesis should be combined with GSI treatments targeting the cancer stem cell population.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3312. doi:1538-7445.AM2012-3312
Abstract 3313: Novel roles of CCN6 in mesenchymal-epithelial transition (MET) and loss of stem cell properties of breast cancer cells
Authors: Huang Wei , Kleer Celina G.
Published: August 2012

Background: Epithelial-mesenchymal transition (EMT) and its reverse process, mesenchymal-epithelial transition (MET), play key roles in regulating embryogenesis. Recent studies have shown that aberrant EMT activation contributes to cancer progression and metastasis. Links between EMT and the gain of stem cell properties have been suggested in physiological and pathological situations. CCN family protein 6 (CCN6/WISP3) is a novel tumor suppressor that regulates diverse cellular functions. Our previous studies have shown the blockade of CCN6 by short hairpin RNA triggers EMT program in benign breast epithelial cells, with decreased expression of epithelial marker E-cadherin and increased activation of corresponding transcriptional repressor, Zeb1 and Snail. Moreover, loss of CCN6 expression also endows benign breast epithelial cells with growth factor-independent properties, such as cell survival and anoikis resistance. However, little is known regarding the functions of CCN6 overexpression in breast cancer progression. Methods: We overexpressed CCN6 in breast cancer cell lines, MDA-MB-231 and SUM-159, which are aggressive, have mesenchymal-like features and low levels of endogenous CCN6. CCN6 provoked functions were studied in vitro by proliferation, motility and invasion assays. Aldehyde dehydrogenase (ALDH1) was used to identify and investigate CCN6 roles in human breast cancer stem cells. Results: We showed that overexpression of CCN6 inhibits cell proliferation, motility and invasion. Importantly, CCN6 overexpression in these cells led to phenotypic changes towards mesenchymal-epithelial transition (MET), characterized by loss of mesenchymal cell marker Vimentin and downregulation of transcriptional factor, Zeb1. CCN6 overexpression significantly reduced the stem cell population of MDA-MB-231 and SUM-159 breast cancer cells. Conclusions: Our data suggest a novel function of CCN6 in regulating MET and reducing the stem cell population of aggressive breast cancer cells.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3313. doi:1538-7445.AM2012-3313
Abstract 3315: Malignant melanoma cells reciprocate aspects of the embryonic neural crest migration program to promote invasion and increased plasticity
Authors: Bailey Caleb M. , Morrison Jason A. , Kulesa Paul M.
Published: August 2012

Melanocytes derive from a highly invasive, multipotent embryonic cell population known as the neural crest. It has been proposed that this ancestral relationship predisposes melanocytes to having metastatic characteristics following neoplastic transformation to melanoma. In support of this hypothesis, we have previously shown that aggressive melanoma cells transplanted into the chick embryonic neural crest microenvironment respond to neural crest signaling mechanisms by emigrating along defined host neural crest pathways. To evaluate whether melanoma cells usurp aspects of the neural crest program typically silenced in differentiated melanocytes, we have employed the chick embryo transplant model coupled with laser capture microdissection to perform an in vivo molecular analysis of more than 70 neural-crest-related genes. Transplanted cells included c8161 aggressive melanoma cells, a poorly aggressive isogenic counterpart termed c81-61, and primary human melanocytes. We demonstrate that all three cell types express many neural-crest-related genes, but that aggressive cells aberrantly express some genes typically lost during melanocyte differentiation. Furthermore, the analysis revealed a significant increase in the ability of aggressive melanoma cells to respond to changing microenvironments by altering gene expression signatures, while transplanted melanocytes appeared to actively suppress these pathways. This increased responsiveness may be attributed to the dynamic regulation of Eph receptor tyrosine kinases and their cognate ephrin ligands, many of which were differentially expressed in aggressive melanoma cells versus poorly aggressive melanoma or primary melanocytes. Thus, metastatic melanoma cells usurp portions of the neural crest program to promote increased invasiveness and plasticity.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3315. doi:1538-7445.AM2012-3315
Abstract 3317: The roles of TMCC3 in breast cancer cells
Authors: Wang Ya- Hui , Yu Alice L.
Published: August 2012

There is growing evidence showing that cancer stem cells are more likely to metastasize and are more frequently detected in the circulation. Thus, it will be important to understand the molecular characteristics of breast cancer stem cells (BCSCs) which may lead to the development of novel targets for CSC-directed therapy. Through phosphoproteomic analysis of BCSC and non-BCSC, we identified differential expression of the transmembrane and coiled-coil domain family 3 (TMCC3). TMCC3 belongs to TEX28 family and is predicted to be an integral membrane protein. The function of TMCC3 is unknown. We found higher expression of TMCC3 in invasive breast cancer cell line and BCSCs than less-invasive breast cancer cell lines, and non-BCSCs, respectively. In addition, TMCC3 expression was greater in distant lymph node metastasis than in primary tumor of human breast cancer xenograft in mouse. Silencing of TMCC3 led to G1 phase arrest with increased apoptotic cells, along with decreased mammosphere formation and ALDH activity which are important features of cancer stem cell. In our previous investigation, the IGF-1R/PI3K/Akt/mTOR pathway was shown to be important for BCSCs survival and maintenance. To explore the possible involvement of TMCC3 in this pathway, we further examined the Akt and mTOR activation in TMCC3-slienced BCSCs. We found the Akt phosphorylation was lost in BCSCs upon TMCC3 knocked down. These findings suggest that TMCC3 is essential for breast cancer cell survival, self-renewal and metastasis. Thus, inhibition of TMCC3 may provide a new therapeutic strategy targeting stem cells.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3317. doi:1538-7445.AM2012-3317
Abstract 332: The role of OLA1, a novel obg-like ATPase, in epithelial-mesenchymal transition
Authors: Bai Li , Rubio Valentina , Mao Renfang , Chen Huarong , Mansour Oula C. , Shi Zheng-Zheng
Published: August 2012

Human OLA1 is a 45 kDa cytosolic protein that belongs to the YchF subfamily of Obg-like P-loop GTPases and is ubiquitously expressed in most tissues. YchF proteins are highly conserved from yeast to humans and all have a greater affinity for ATP than GTP. Based on structure analysis, OLA1 is predicted to be a regulatory protein that interacts with its client protein(s) and exerts its downstream functions by the conformational switch between the ADP- and ATP-bound forms. However, the actual physiological functions of OLA1 are poorly understood. Our earlier studies have suggested that OLA1 is a negative regulator of cellular antioxidant response and functions through post-transcriptional mechanisms (PNAS 2009; 106:15356). Oxidative stress, mediated by reactive oxygen species (ROS), has been implicated in epithelial-mesenchymal transition (EMT), a fundamental phenotypic alteration closely associated with cancer progression and metastasis. In this study we investigated the effect of OLA1 on EMT and the possible underlying mechanisms. We first demonstrated that the human lung epithelial carcinoma A549 cells, after exposure to transforming growth factor (TGF)-β1 for 3 days, underwent EMT as evidenced by acquisition of a mesenchymal-like morphology, down-regulation of epithelial-specific marker E-cadherin, and induction of mesenchymal marker vimentin. However, knockdown of OLA1 with sequence-specific siRNA in A549 cells resulted in a significant attenuation of the TGF-β1-induced EMT phenotypes, including those in cellular morphology and the EMT markers E-cadherin and vimentin, as compared with the cells transfected with the control non-targeting siRNA. Next, we questioned if this inhibitory effect on EMT was through inhibiting the Akt/GSK3β/Snail pathway. When treated with TGF-β1, OLA1-knockdown cells showed impaired GSK-3β phosphorylation (indicating an overactivation of GSK-3β), and subsequently reduced abundance of the transcription factor Snail that functions as the suppressor of E-cadherin gene expression. Concurrently, OLA1-knockdown cells exhibited significantly decreased TGF-β1-mediated intracellular ROS production assessed by DCFDA staining and flow cytometry analysis. Furthermore, we found that treatment of H2O2 abrogated resistance of OLA1-knockdown cells to TGF-β1-induced EMT, evidenced by phosphorylation of GSK-3β, upregulation of Snail and changes in expression of EMT markers. These results suggest that OLA1 plays an important regulatory role in EMT of human cancer cells, and inhibition of OLA1 may have a negative effect on EMT, probably through down-regulation of the ROS-dependent TGF-β1 signaling pathway.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 332. doi:1538-7445.AM2012-332
Abstract 3322: Phenotypic plasticity in the normal breast
Authors: Sauder Candice Anne Marcum , Koziel Jillian E. , Choi MiRan , Fox Melanie J. , Badve Sunil , Blosser Rachel J. , Mathieson Theresa , Rufenbarger Constance A. , Henry Jill E. , Storniolo Anna Maria V. , Herbert Brittney-Shea , Clare Susan E.
Published: August 2012

Metaplasia is observed in almost all epithelial cancers. The origins of metaplasia are obscure although chronic inflammation is thought to be one etiology. We present data that suggests that an origin of metaplasia is the normal resident stem cell population. Methods: Starting from 10 gauge tissue cores of normal breast donated to the Komen Tissue Bank, 28 normal mammary epithelial (HME) and 33 normal stromal (HMS) cell lines were established using an organoid isolation method after digestion with enzymes for 24 hours. The HME cell lines were characterized by immunohistochemistry (IHC). Ploidy was assayed. Cellular morphology was observed both on two-dimensional and in three-dimensional culture systems. The HME cells were subjected to FACS analysis using multiple antibodies. Results: 96.9% of early passage cells are diploid. The HME cells express vimentin, CK 5/6, p63, CD 10, CK 18, and HER-1 when grown on two dimensional plastic surfaces. Cells placed in the center of a sandwich of Matrigel uniformly make spheres 37mm-325mm in diameter. Hematoxylin and eosin staining of the formalin-fixed and paraffin-embedded sections of these spheres reveal keratinized squamous differentiation. When the cells are grown on Laminin, Collagen Type IV, or Fibronectin surfaces multiple cell types are observed including osteoclasts, distinguished by the presence of Tartrate Resistant Acid Phospatase; and chondrocytes, confirmed by staining with Alcian Blue. Other cells with a spindle-shape and cytoplasmic vacuoles turn a dark reddish-brown color when stained with Oil Red O, characteristics of adipocytes. In other areas of the culture, the cells form a syncitium and they express the protein MyoD, a marker of immature muscle. Finally, there are numerous cells with long, dendritic processes. These cells express Nestin, glial fibrillary acidic protein (GFAP), and beta-III tubulin. Using FACS, the HME cells were found to be CD49f positive and EpCAM negative. Multiple nucleoli were confirmed using anti-Nucleostemin IHC. Conclusions: Phenotypic plasticity is common to all the HME cell lines characterized to date. Differentiation into cells of mesodermal and ectodermal origin, CD49+/EpCAM- by FACS, and the presence of multiple nucleoli suggest that the isolated cells are a multipotent/stem cell residing in the normal adult breast. These cells, through a series of yet to be elucidated events, may be the cells of origin of both benign and malignant metaplasia observed in breast lesions.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3322. doi:1538-7445.AM2012-3322
Abstract 3323: Hyaluronan synthase 2 promotes breast cancer bone metastasis by stimulating interaction of cancer stem-like cells with tumor-associated macrophage
Authors: Okuda Hiroshi , Xing Fei , Kobayashi Aya , Watabe Misako , Pai Sudha K. , Liu Wen , Pandey Puspa R. , Fukuda Koji , Iiizumi Megumi
Published: August 2012

The molecular mechanism of metastatic progression, particularly the roles of microenvironment of the metastasized organs, is as yet poorly understood. In this study, we have isolated cancer stem cell-like cells (CSC) from human breast cancer cell lines, MDA-MB231, MDA231BoM and MDA231BrM. CSC were isolated from each line using surface markers (CD24-, CD44+, ESA+) and their tumor initiating abilities were tested in animal. We then performed an analysis of global expression profile for these CSCs using the Affymetrix expression array and found that among all up-regulated genes, two genes (HAS2 and MMP1) were significantly correlated with overall- and metastasis-free survival when they were cross-examined in GEO database. To further examine the role of HAS2 in tumor metastasis in vivo, we prepared CSC from MDA231BoM with or without carrying a shRNA expression vector for HAS2. The CSC were then implanted into nude mice by intra-cardiac injection followed by oral administration of 4MU, a specific inhibitor of HAS genes. We found that the injection of CSC of MDA231BoM which expresses shRNA to HAS2 significantly improved the survival rate of animals. In addition, administration of 4MU significantly suppressed tumor metastasis of CSC, suggesting that inhibition of HAS2 can indeed reduce the incidence of metastasis in vivo. We also examined the effect of tumor associated macrophage (TAM) on growth of CSC in animals by injecting CSC of MDA231-BoM with or without TAM into the tibial bone. We found that co-injection of TAM with CSC significantly augmented the growth of tumor in their legs. Moreover, the interaction between cancer cell and TAMs enhanced secretion of PDGF-BB from TAMs which then activated stromal cells and in turn enhanced self-renewal of CSCs by secretion of FGF9 and FGF7. We also found that treatment of bone marrow stem cell (BMSC) with PDGF followed by co-culture of CSC significantly promoted proliferation of CSC. Collectively, our results suggest that TAM and BMSC promote self-renewal of CSC in bone microenvironment via direct interaction between hyaluronan and CD44. Our results also provide strong rationale to target HAS2 by using 4MU in order to block the metastatic progression of CSC in breast cancer.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3323. doi:1538-7445.AM2012-3323