bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 1876: Cell cycle-dependent front polarized cell migration requires Aurora kinase A
Authors: Chu Tony Lok Heng , Zhou Lixin , Won Jennifer , Mohan Pooja , Nemirovsky Oksana , Fotovati Abbas , Nielsen Torsten , Pante Nelly , Maxwell Christopher
Published: July 2017

PROBLEM- Metastasis accounts for 90% of deaths from carcinomas.

BACKGROUND- To initiate migration, epithelial cells must front-rear polarize and reorganize the microtubule cytoskeleton directed toward the lamillipodia or leading edge. During mitosis, centrosome maturation, duplication, spindle pole assembly and chromosome segregation require Aurora kinase A (AURKA), which is optimally active when complexed with TPX2. Accumulating evidences identify non-mitotic functions for AURKA, such as cell migration and ciliary reorganization. Overexpression of AURKA leads to tumorigenesis in breast cancer. Mechanisms that activate non-mitotic AURKA and promote cell migration as well as metastasis are yet to be uncovered.

RESULTS- In a large breast cancer tissue microarray (n= 3,175), the abundance of phosphorylated RHAMM (a substrate of AURKA) predicted overall and relapse-free survival in ER-negative, basal-subtype, and triple negative breast tumors. We studied the AURKA-TPX2-RHAMM signaling axis in mammary (MCF10A and nMUMG-FUCCI) and HeLa cells. In scratch wound assays, migratory cells were found to have front-polarized centrosomes, which correlated with G2 cell cycle phase, phosphorylated AURKA and elevated microtubule nucleation at centrosomes. Small molecule inhibition of Aurora kinase activity impaired centrosome polarity and retarded the kinetics of cell migration. In parallel, silencing RHAMM altered TPX2 location as indicated by immunofluorescence and immuno-EM, which located TPX2 to the nuclear envelope and nuclear pore basket. Nuclear import of recombinant TPX2 was attenuated in RHAMM silenced cells and nuclear envelope accumulation was elevated.

CONCLUSION- The AURKA-TPX2-RHAMM axis regulates centrosome polarity, microtubule nucleation and directional cell migration while phosphorylated RHAMM predicts survival in aggressive forms of breast cancer.

Citation Format: Tony Lok Heng Chu, Lixin Zhou, Jennifer Won, Pooja Mohan, Oksana Nemirovsky, Abbas Fotovati, Torsten Nielsen, Nelly Pante, Christopher Maxwell. Cell cycle-dependent front polarized cell migration requires Aurora kinase A [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1876. doi:10.1158/1538-7445.AM2017-1876
Abstract 189: Development of peptidomimetic inhibitors of the ERG gene fusion product in prostate cancer
Authors: Wang Xiaoju
Published: July 2017

Transcription factors play a key role in the development of diverse cancers, and therapeutically targeting them has remained a challenge. In prostate cancer, the gene encoding the transcription factor ERG is recurrently rearranged and plays a critical role in prostate oncogenesis. Here, we identified a series of peptides that interact specifically with the DNA binding domain of ERG. ERG inhibitory peptides and derived peptidomimetics (EIPs) bound with high affinity and specificity leading to proteolytic degradation of ERG protein. The EIPs attenuated ERG-mediated transcription, chromatin recruitment, protein-protein interactions, cell invasion, and tumor growth. Thus, peptidomimetic targeting of transcription factor fusion products may provide a promising therapeutic strategy for prostate cancer as well as other cancers.

Citation Format: Xiaoju Wang. Development of peptidomimetic inhibitors of the ERG gene fusion product in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 189. doi:10.1158/1538-7445.AM2017-189
Abstract 1896: Soluble lung-derived selectins promote breast cancer cell migration
Authors: Khan Sami U. , Chu Jenny E. , Xia Ying , Allan Alison L.
Published: July 2017

Ninety percent of all breast cancer-related deaths are attributed to metastasis, as current therapies are largely non-curative in the metastatic setting. One of the most common sites of breast cancer metastasis is the lung. Previous work from our lab has demonstrated that there are over 70 soluble factors produced by murine lung tissue, many of which have been associated with cancer cell growth, migration, invasion and metastasis. However, the relative contribution of each individual factor in enhancing the metastatic behavior of breast cancer cells has not been examined in detail. Three of the identified soluble lung-derived proteins are E-, L- and P-selectin, and these were the focus of the current study. The selectins are a family of glycoproteins with known physiological roles in vascular/immune cell migration, and soluble selectins have been clinically observed to be elevated in the sera of cancer patients. We hypothesized that soluble lung-derived selectins enhance the metastatic behavior of breast cancer cells. We used a 2D ex vivo model of the soluble lung microenvironment, generated by isolating conditioned media from dissociated healthy murine lung. E-, L- and P-selectin were then individually immunodepleted from the lung-conditioned media (lung-CM) and the subsequent functional effect on breast cancer cell migration and proliferation was assessed using transwell migration and BrdU incorporation assays, respectively. MDA-MB-231 and SUM149PT human breast cancer cells demonstrated significantly enhanced cell migration toward lung-CM relative to basal media, and this migration was significantly reduced when E-, L- or P-selectin were depleted (p<0.05). In contrast, although MDA-MB-231 and SUM149PT human breast cancer cells demonstrated significantly enhanced cell proliferation in response to lung-CM relative to basal media (p<0.05), depletion of soluble E-, L- or P-selectin from lung-CM had no influence on reducing proliferation (p>0.05). Ongoing studies are aimed at elucidating the pathways through which soluble lung-derived selectins exert their pro-migratory function. If these results implicate a common receptor and downstream signalling pathways for all three selectins, these ligand-receptor interactions may have value as potential therapeutic targets for the prevention of pulmonary metastasis in breast cancer patients.

Citation Format: Sami U. Khan, Jenny E. Chu, Ying Xia, Alison L. Allan. Soluble lung-derived selectins promote breast cancer cell migration [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1896. doi:10.1158/1538-7445.AM2017-1896
Abstract 1897: The role of nuclear deformation and rupture in breast cancer cell migration
Authors: McGregor Alexandra L. , Elacqua Joshua J. , Bell Emily S. , Lammerding Jan
Published: July 2017

Invasion of tumor cells into the surrounding tissue is a critical step in the metastatic cascade, which is responsible for the vast majority of cancer-related deaths. Recent findings have begun to demonstrate that the biophysical properties of the cell nucleus play a critical role in this process. During invasion, cancer cells must often pass through interstitial spaces much smaller than the cell nucleus. While the cell cytoplasm easily maneuvers through confined spaces, the deformability of the nucleus, which is the largest and stiffest organelle, ultimately determines how efficiently a cell can pass through a tight space. Furthermore, as the nucleus deforms to move through a confined space, it can exhibit transient loss of nuclear envelope (NE) integrity, where the nuclear membranes rupture and nuclear and cytoplasmic components interchange. We previously showed that chromatin is exposed to the cell cytoplasm during NE rupture, and cells can incur DNA damage. While this work revealed that cancer cell migration requires nuclear deformation and can involve NE rupture, it is currently unknown whether such events correlate with metastatic potential. We used a panel of human breast cancer cells lines, including claudin-low, basal-like, and lumen-like cells. Cells were stably labeled with a green fluorescent protein fused to a nuclear localization sequence (NLS-copGFP) to investigate whether some cell lines were particularly well suited to migrate through tight spaces, and if any were particularly prone to NE rupture. The cells were loaded into microfluidic devices that were designed to mimic interstitial in vivo spacing and monitored by time-lapse microscopy. Devices contained constrictions 5 µm tall and 1, 2, or 15 µm wide. We found that claudin-low cell lines such as BT-549 migrated through confined spaces (1+2 µm wide constrictions) significantly faster and more efficiently than basal-like HCC70 cancer cells. In contrast, the various cell lines displayed similar migration rates in the 15 µm wide constrictions, which do not require nuclear deformation. Thus, the differences observed between the cell lines reflect specific defects in migration through confined spaces, and not general migration defects. We are currently expanding our studies to a larger panel of cancer cell lines and to mouse breast cancer models. If confirmed, these results could indicate that highly invasive cancer cells may benefit from more deformable nuclei that facilitate movement through tight spaces encountered in the tissue microenvironment.

Citation Format: Alexandra L. McGregor, Joshua J. Elacqua, Emily S. Bell, Jan Lammerding. The role of nuclear deformation and rupture in breast cancer cell migration [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1897. doi:10.1158/1538-7445.AM2017-1897
Abstract 1917: AXL inhibition in ovarian cancer stem-like cells increases chemo-sensitivity
Authors: Kim Jin-Young , Chung Hyewon , Shin So-Jin , Ha Eunyoung , Cho Chi-Heum
Published: July 2017

Cancer stem-like cells (CSCs), a small population of cancer cells with regenerative potential, may lead to the initiation and progression of secondary tumors. Long-term chemotherapeutic drug treatment can also result in the generation of a drug resistant population of cancer stem cells. In this study we have isolated CD44+ and CD117+ enriched ovarian cancer stem-like cells from primary cultured ascites cells removed from ascites fluid of naïve ovarian cancer patients. AXL, a type I receptor tyrosine kinase family member, is found to be over-expressed in ovarian cancer stem-like cells. Cell viability assays showed that ovarian CSCs are chemo-resistant to concentration dependent paclitaxel treatment. AXL was inhibited using a R428 and cell viability assays and apoptotic assays in the presence of paclitaxel were performed, which made the CSCs chemo sensitive. Si-RNA based inhibition of AXL also displayed a similar pattern of improving the sensitivity of the stem-like cells to the chemotherapeutic drug. PI3-kinase/Akt signaling pathway has also been shown to be involved in decreasing the cell viability during the inhibition of AXL. In conclusion, this study establishes the role of AXL, a receptor tyrosine kinase, as one of the key proteins influencing the chemo resistance in ovarian cancer stem-like cells.

Citation Format: Jin-Young Kim, Hyewon Chung, So-Jin Shin, Eunyoung Ha, Chi-Heum Cho. AXL inhibition in ovarian cancer stem-like cells increases chemo-sensitivity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1917. doi:10.1158/1538-7445.AM2017-1917
Abstract 192: Targeting stearoyl CoA desaturase 1 (SCD1) in hepatobilliary carcinoma
Authors: Copland John Alton , Marlow Laura A. , Bok Ilah , Miller James L. , Akiko Matsuda , Asmann Yan W. , Sarangi Vivekananda , Alberts Steven R. , Mody Kabir , Roberts Lewis R. , Truty Mark J. , Patel Tushar C.
Published: July 2017

Background: The paucity of effective therapeutic agents for hepatocellular cancer (HCC) underscores the critical need for more effective therapeutic strategies. Recent studies indicate lipid biosynthesis and desaturation is required for HCC survival. Targeting these may prove beneficial because such changes contribute to therapeutic resistance. Stearoyl CoA desaturase (SCD1), a key mediator of fatty acid (FA) biosynthesis and rate-limiting in conversion of saturated fatty acids (SFAs) to mono-unsaturated fatty acids (MUFAs), is upregulated in HCC and many other cancers. As such, we therapeutically targeted a novel lipogenic tumor survival mechanism mediated by SCD1 as a means to combat the chemoresistance associated with HCC. In so doing, we evaluated a novel lead SCD1 inhibitor in HCC.

Methods: Paraffin embedded patient HCC tissues were examined for SCD1 expression. Using combined computational and synthetic chemistry approaches, we synthesized four novel specific SCD1 inhibitors with SSI-4 being the lead SCD1 inhibitor. HCC cell lines were examined using proliferation assays for response to SSI-4. IC50 concentrations for blocking SCD1 enzyme activity was determined. Blood half-life and bioavailability of single dose SSI-4 was determined. Mechanisms of action of SCD1 were examined that included Endoplasmic reticulum (ER) stress. In vivo, antitumor activity was determined using HCC patient derived xenograft (PDX) mouse models.

Results: We identified elevated SCD1 mRNA and protein in HCCs tissues. SSI-4 dose-dependently inhibits cell proliferation in HCC cell lines with specificity demonstrated by oleic acid (MUFA) co-culture. Single dose oral gavage SSI-4 demonstrated a half-life of ~4 hours and excellent oral bioavailability. SSI-4 was well tolerated with long-term daily dosing. SSI-4 treatment of HCC cells and tumors led to endoplasmic reticulum (ER) stress followed by apoptotic cell death. Single agent SSI-4 demonstrated antitumor activity in HCC PDX mouse models with suppression of ER stress regulated proteins.

Conclusions: Targeting a novel lipid metabolic pathway in HCC may provide effective therapy for aggressive HCC.

Citation Format: John Alton Copland, Laura A. Marlow, Ilah Bok, James L. Miller, Matsuda Akiko, Yan W. Asmann, Vivekananda Sarangi, Steven R. Alberts, Kabir Mody, Lewis R. Roberts, Mark J. Truty, Tushar C. Patel. Targeting stearoyl CoA desaturase 1 (SCD1) in hepatobilliary carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 192. doi:10.1158/1538-7445.AM2017-192
Abstract 1921: Stem-like clusters of CD44+ circulating tumor cells seed metastases in breast cancer
Authors: Liu Xia , Chen Wenjing , Taftaf Rokana , Liu Huiping
Published: July 2017

Background: Circulating tumor cells (CTCs) are cells shed from primary tumor and circulate in the peripheral blood. They are considered the seeds of metastasis. Compared to single CTCs, clusters of multiple CTCs possess 50 times higher metastatic capacity in mouse breast cancer models. However, the mechanisms underlying the metastatic promotion effect of CTC clusters are unclear. In addition, whether clustered CTCs have cancer stem cell (CSC) properties and what stem cell markers they express have not been determined.

Methods: Immunohistochemistry (IHC) was used to detect CTCs in vascular structures, and the stem cell markers in single and clustered CTCs. Lung metastases were either monitored by bioluminescence imaging (BLI) or visualized by fluorescence microscopy. Stem cell properties were examined by mammosphere assays in vitro and tumorigenic assays in vivo. Clustering assay was performed by culturing cells in Poly-HEMA coated plates (for cell lines) or collagen-coated plates (for primary cells derived from patient-derived breast tumor xenografts, PDXs), and then monitored by IncuCyte live cell dynamic imaging.

Results: CD44+ enriched circulating tumor cell (CTC) clusters were found in the lung/liver vascular structures in vivo in both metastatic breast cancer patients and PDXs that develop spontaneous lung metastases. Comparing to single CD44+ cells, clustered CD44+ cells formed more mammospheres, increased tumorigenic potential, and promoted lung colonization. Combining siRNA-mediated knockdown and CRISPR/Cas-based knockdout, we found that CD44 is required for breast tumor cell cluster formation and lung colonization upon tail vein seeding. During cluster formation, EGFR was activated, which improved both stemness and survival of clustered CTCs. Anti-EGFR antibody mimicked CD44 knockdown to inhibit cluster formation of PDX-derived tumor cells. Administration of EGFR inhibitor Erlotinib efficiently inhibited CD44+ cells-mediated spontaneous metastases to the lungs without affecting primary tumor growth.

Conclusions: Our data provide new insights into the cellular and molecular mechanisms of stem-like clustered CD44+ cells-seeded metastasis, and implicate that targeting CD44+ CTC clusters by inhibition of EGFR activity could be a new therapeutic strategy to treat metastatic breast cancer.

Citation Format: Xia Liu, Wenjing Chen, Rokana Taftaf, Huiping Liu. Stem-like clusters of CD44+ circulating tumor cells seed metastases in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1921. doi:10.1158/1538-7445.AM2017-1921
Abstract 1924: Caveolin-1 inhibits mammary carcinogenesis via suppressing c-myc-induced metabolism reprogramming in breast cancer stem cells
Authors: Wang Zhiyu , Wang Neng , Wang Shengqi , Zheng Yifeng
Published: July 2017

Purpose: Caveolin-1(CAV1), a membrane constituent protein, exhibits tumor suppressor activities in multiple malignancies, whereas the underlying mechanisms remain unclear. The current study aimed to determine the significance of CAV-1 in regulating cancer metabolism and its relation to breast cancer stem cells (CSCs).

Experimental Design: The anti-carcinogenic function of CAV1 was evaluated by in vitro cell model, CAV1 knockout mice and MMTV-PyMT spontaneous breast cancer xenografts. Glycolysis activity and mitochondrial metabolism were assessed by immunoblotting, oxygen consumption and mitotacker staining analysis. Mammosphere formation, tumorigenic ability, reattachment differentiation and signaling pathway analysis were applied to study the regulation effects of CAV1 on breast CSCs. The clinical significance of CAV1 was also analyzed by human tissue microarray.

Results: In both mammary transformed cells and spontaneous breast cancer xenografts, CAV1 is found significantly downregulated and positively correlated to increased glycolysis activity and impaired mitochondrial metabolism. Cav1 knockout results in increased ductal hyperplasia, associating with increased stem cell population and glycolysis metabolism. Breast CSCs exhibits glycolytic phenotype and decreased CAV1 expression compared to normal mammary stem cells, and CAV1 overexpression significantly limits CSCs’ self-renewal via inhibiting c-myc induced glycolysis. Clinical investigation suggests that high CAV1 expression is revealed with better overall survival and decreased CSCs population.

Conclusions: CAV1 loss facilitates mammary carcinogenesis via enhancing glycolytic activity in breast CSCs, and CAV1 based therapy might become a novel strategy for breast cancer prevention.

Note: This abstract was not presented at the meeting.

Citation Format: Zhiyu Wang, Neng Wang, Shengqi Wang, Yifeng Zheng. Caveolin-1 inhibits mammary carcinogenesis via suppressing c-myc-induced metabolism reprogramming in breast cancer stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1924. doi:10.1158/1538-7445.AM2017-1924
Abstract 1927: The significance of calreticulin in pancreatic cancer: a molecule highly expressed in pancreatic cancer stem-like cells
Authors: Matsukuma Satoshi , Yoshimura Kiyoshi , Oga Atsunori , Inoue Moeko , Fujimtoto Takuya , Kuramasu Atsuo , Fuse Masanori , Tsunedomi Ryouichi , Eguchi Hidetoshi , Matsui Hiroto , Kanekiyo Shinsuke , Tokumitsu Yukio , Tomochika Shinobu , Iida Michihisa , Tokuhisa Yoshihiro , Sakamoto Kazuhiko , Suzuki Nobuaki , Furuya-Kondo Tomoko , Itoh Hiroshi , Takeda Shigeru , Yamamoto Shigeru , Yoshino Shigefumi , Hazama Shoichi , Ueno Tomio , Nagano Hiroaki
Published: July 2017

Cancer stem-like cells (CSLCs) in solid tumors are thought to be resistant to conventional chemotherapy or molecular targeting therapy and to contribute to cancer recurrence and metastasis. In this study, we aimed to identify a biomarker of pancreatic CSLCs (P-CSLCs). P-CSLC-enriched population was generated from pancreatic cancer cell lines using our previously reported method and its protein expression profile was compared with that of parental cells by two-dimensional electrophoresis and tandem mass spectrometry. The results indicated that a chaperone protein calreticulin (CRT) was significantly upregulated in P-CSLCs compared to parental cells. Flow cytometry analysis demonstrated that CRT was mostly localized to the surface of P-CSLCs and did not correlate with the levels of CD44v9, another P-CSLC biomarker. Furthermore, the side population in CRThigh/CD44v9low population is much higher than that in CRTlow/CD44v9high population. CRT expression was also assessed by immunohistochemistry in pancreatic cancer tissues (n = 80) obtained after radical resection and was found to be associated with patients’ clinicopathological features and disease outcomes in the Cox’s proportional hazard regression model. Multivariate analysis identified CRT as an independent prognostic factor for pancreatic cancer patients, along with age and post-operative therapy. Our results suggest that CRT can serve as a biomarker of P-CSLCs and a prognostic factor associated with poorer survival of pancreatic cancer patients. This novel biomarker can be useful for detecting P-CSLCs independently, which had been detectable by multiple surface markers like CD24, CD44 and ESA. We will present CSLCs properties of CRThigh population in P-CSLCs.

Citation Format: Satoshi Matsukuma, Kiyoshi Yoshimura, Atsunori Oga, Moeko Inoue, Takuya Fujimtoto, Atsuo Kuramasu, Masanori Fuse, Ryouichi Tsunedomi, Hidetoshi Eguchi, Hiroto Matsui, Shinsuke Kanekiyo, Yukio Tokumitsu, Shinobu Tomochika, Michihisa Iida, Yoshihiro Tokuhisa, Kazuhiko Sakamoto, Nobuaki Suzuki, Tomoko Furuya-Kondo, Hiroshi Itoh, Shigeru Takeda, Shigeru Yamamoto, Shigefumi Yoshino, Shoichi Hazama, Tomio Ueno, Hiroaki Nagano. The significance of calreticulin in pancreatic cancer: a molecule highly expressed in pancreatic cancer stem-like cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1927. doi:10.1158/1538-7445.AM2017-1927
Abstract 1928: The lung microenvironment promotes stem-like phenotype and behavior of breast cancer cells
Authors: Sadri Ashkan , Allan Alison L.
Published: July 2017

Ninety percent of breast cancer-related mortalities result from metastasis, a process whereby the primary tumour disseminates and targets distant secondary organs. We have previously demonstrated that stem-like breast cancer cells expressing high levels of aldehyde dehydrogenase (ALDH) and CD44 are critical for metastasis. These ALDHhiCD44+ cells preferentially migrate and/or metastasize to the lung and bone microenvironments, where secondary tumours can severely impede organ function. However, the specific role of the lung and bone marrow (BM) microenvironments in supporting and/or promoting metastasis of ALDHhiCD44+ cells remains poorly understood, and this is the purpose of the current study. Using a 2D ex vivo model, lung and bone marrow (BM) conditioned media (CM) enriched in tissue-specific soluble proteins were generated from murine tissues and utilized in cell culture to assess their ability to influence the stem-like phenotype and behavior of MDA-MB-468 human breast cancer cells. Exposure to lung-CM promoted the generation of a viable, non-adherent (“floating”) breast cancer cell subpopulation that was not observed in response to BM-CM or basal media (p<0.05). Phenotypic analysis by flow cytometry and gene expression analysis by RT-qPCR revealed notable differences in ALDH/CD44 phenotype in whole cell populations, adherent subpopulations, and/or non-adherent subpopulations in response to 72 hour exposure to lung-CM relative to basal media. In particular, the non-adherent breast cancer cell subpopulation generated in response to lung-CM demonstrated increased CD44 and ALDH1A3 gene expression and decreased ALDH1A1 gene expression relative to the adherent subpopulation (p<0.05). No significant phenotypic or gene expression differences were observed in MDA-MB-468 breast cancer cells in response to BM-CM. Taken together with our previous studies, these findings suggest that while both the lung and bone microenvironments support breast cancer cells with a pre-existing ALDHhiCD44+ phenotype, the lung microenvironment may additionally help to even further promote stem-like phenotype and behavior in the secondary metastatic site. Ongoing studies are aimed at elucidating the mechanisms by which aggressive ALDHhiCD44+ breast cancer cells interact with and/or are influenced by the lung microenvironment during metastatic progression.

Citation Format: Ashkan Sadri, Alison L. Allan. The lung microenvironment promotes stem-like phenotype and behavior of breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1928. doi:10.1158/1538-7445.AM2017-1928
Abstract 1929: A novel orthotopic mouse model in sarcomas
Authors: Monclús Silvia Garcia , Martínez Juan Huertas , Tena Laura Lagares , Varona Santiago Rello , Rabaiget Olga Almacellas , Martin David Herrero , Alemany Roser López , Tirado Oscar Martinez
Published: July 2017

Pediatric sarcomas are a heterogeneous group of bone and soft tissue malignancies affecting children and young adults. One of the most important prognostic factors of those diseases is the presence of metastasis at diagnosis. In that context, we have developed a novel orthotopic model which consists in injecting Ewing Sarcoma (ES) or Rhabdomyosarcoma (RMS) tumor cells at the gastrocnemius muscle of mice and extracting the tumor at a of 10 mm x 13 mm volume. After surgery, animals maintain a complete functional extremity and can live until lung metastasis detection (about 60 days post-injection).

Moreover, we have validated the suitability of the model with an Ewing Sarcoma EphA2-low expression cell line. Epha2 is a tyrosine kinase receptor that has been found overexpressed in a wide variety of tumors and correlated with malignant phenotype. In this study we report that EphA2 receptor is phosphorylated at S897 in a panel of ES cell lines, which is related to the oncogenic properties of the receptor. Stable silencing of EphA2 in two different ES cell lines resulted in a decrease in the clonogenic, proliferation, migration and invasion capacity in vitro. Moreover, we performed an experimental metastasis assay, injecting tumor cells through the tail vain of mice and observed a reduction in the lung metastasis incidence in EphA2 silenced cells. We then used this new orthotopic metastasis model to validate the impairment in the invasion capacity of EphA2 silenced cells and confirm the decrease in lung metastasis incidence, indicating an oncogenic role for EphA2 in ES. This novel orthotopic metastasis model is a valuable tool both for the study of spontaneous metastasis and also for evaluating therapeutic index in the onset of metastasis, which can also be applied to the study of other pedriatric sarcomas.

Citation Format: Silvia Garcia Monclús, Juan Huertas Martínez, Laura Lagares Tena, Santiago Rello Varona, Olga Almacellas Rabaiget, David Herrero Martin, Roser López Alemany, Oscar Martinez Tirado. A novel orthotopic mouse model in sarcomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1929. doi:10.1158/1538-7445.AM2017-1929
Abstract 1937: Evolution of neuroblastoma patient-derived orthotopic xenografts through space and time
Authors: Braekeveldt Noémie , Fransson Susanne , Stedingk Kristoffer von , Öra Ingrid , Noguera Rosa , Martinsson Tommy , Nord David Gisselsson , Påhlman Sven , Bexell Daniel
Published: July 2017

Background Recently, we established orthotopic neuroblastoma patient-derived xenografts (PDXs) which maintain the phenotypic, genomic, and stromal hallmarks of patient tumors. Here we examined how PDXs evolve following years of in vivo growth.

Materials and Methods We established up to eight in vivo generations of neuroblastoma orthotopic PDXs through serial passaging in NSG mice. RNA sequencing, exome sequencing and SNP array analysis were used to analyze patient tumors and PDXs from different in vivo generations.

Results Using SNP analysis, we found mostly a remarkable genomic stability at chromosomal level between patient tumors, early and late PDX generations. RNA-seq revealed that patient tumors expressed higher levels of genes involved in immune responses and ECM metabolism compared to PDXs. Different PDX samples clustered correctly into their respective tumor type. PDXs from all early generations did not separate from PDXs from late generations. Thus, gene expression levels are surprisingly often quite stable despite years of in vivo growth. To shed light on neuroblastoma intratumor heterogeneity, we implanted 10 different tumor fragments from a single patient tumor into mice. We classified the 10 mice into three groups based on the time periods required for tumor growth. RNA-seq showed that each of these groups had a distinct gene expression profile and pathways involved in neuroblastoma progression have been identified.

Conclusions Neuroblastoma orthotopic PDXs are often very stable at chromosomal and gene expression levels despite years of in vivo growth. We utilized multiple PDXs to show functional intratumor heterogeneity coupled to distinct gene expression profile.

Citation Format: Noémie Braekeveldt, Susanne Fransson, Kristoffer von Stedingk, Ingrid Öra, Rosa Noguera, Tommy Martinsson, David Gisselsson Nord, Sven Påhlman, Daniel Bexell. Evolution of neuroblastoma patient-derived orthotopic xenografts through space and time [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1937. doi:10.1158/1538-7445.AM2017-1937
Abstract 1938: Exportin-1 (XPO1) is a novel therapeutic biomarker for patients with neuroblastoma
Authors: Galinski Basia , Luxemburg Marcus , Ewart Michelle , Landesman Yosef , Weiser Daniel
Published: July 2017

Background: Half of patients with high-risk neuroblastoma succumb to disease, yet these patients with inferior outcome cannot be identified at diagnosis despite contemporary risk stratification that integrates MYCN copy number status, tumor histology, and patient age. We have shown that overexpression of Exportin-1 (XPO1) is associated with poor survival in neuroblastoma, affirming what has been identified across a range of malignancies. Selinexor (KPT-330, Karyopharm Therapeutics), an XPO1 inhibitor in early phase clinical trials, inhibits the nuclear to cytoplasmic translocation of tumor suppressor and growth regulatory proteins implicated in oncogenesis. We hypothesized that protein expression level of XPO1 in neuroblastoma cell lines predict the effective response of treatment with selinexor.

Methods: We used a panel of early passage patient derived neuroblastoma cell lines including IMR5, NLF, KELLY, NB-EBC1, and SKNSH that are representative of the spectrum and genetic diversity of human disease. Cell lines were treated with varying concentrations of selinexor. RNA and whole cell protein extract as well as nuclear and cytoplasmic fractionated protein was obtained from untreated and treated conditions. Treated and untreated cells were also pelleted, and, along with patient tumor samples, formalin-fixed and paraffin-embedded for microscopic and immunohistochemical (IHC) evaluation.

Results: Compared to noncancerous cell lines, neuroblastoma cell lines have high protein and mRNA expression of XPO1; higher expression correlates with greater sensitivity to selinexor. Selinexor treatment reduced XPO1 protein and increased XPO1 mRNA expression, and it lead to nuclear retention of XPO1 cargo such as p53 and survivin. Comparison of microscopic features and protein staining intensity of patient samples with known XPO1 expression is ongoing.

Conclusions: This study provides rationale for XPO1 protein abundance as a potential biomarker of therapeutic response to the drug selinexor. By using patient derived neuroblastoma cell lines, we are able to evaluate the response of selinexor and XPO1 expression in the context of this enigmatic disease. These data suggest that initial drug response to selinexor can be predicted early in the course of disease and more investigation of the high expression of XPO1 seen in patients with poor outcome will provide strategies for combinatorial treatment approaches.

Citation Format: Basia Galinski, Marcus Luxemburg, Michelle Ewart, Yosef Landesman, Daniel Weiser. Exportin-1 (XPO1) is a novel therapeutic biomarker for patients with neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1938. doi:10.1158/1538-7445.AM2017-1938
Abstract 1939: Loss of MST/Hippo signaling promotes tumorigenesis in a genetically engineered mouse model of fusion-positive alveolar rhabdomyosarcoma
Authors: Oristian Kristianne , Crose Lisa E. , Bentley Rex , Kuprasertkul Nina , Kirsch David G. , Linardic Corinne
Published: July 2017

While improvement in survival for pediatric cancer patients over the last 40 years has been encouraging, certain cancer types evade cure. One such example is fusion-positive alveolar rhabdomyosarcoma (aRMS), a pediatric soft tissue sarcoma of mesenchymal origin with skeletal muscle features and a 5-year survival rate of <50%. A hallmark of this aggressive malignancy is the t{2;13} translocation fusion gene PAX3-FOXO1 (PF). Primary cell-based modeling experiments in our laboratory and others have shown that PF is necessary but not sufficient for aRMS tumorigenesis, indicating additional molecular alterations are required to initiate and sustain tumor growth. Previously we showed that PF-positive aRMS tumorigenesis relies on alterations in Hippo pathway signaling, including upregulation of the YAP transcriptional co-activator, and downregulation of the MST/Hippo kinase, which occurs when the scaffold protein RASSF4 (itself a PF transcriptional target) binds and inhibits MST1/Hippo. We hypothesized that ablating MST/Hippo signaling in an existing genetically engineered mouse model (GEMM) of aRMS would accelerate tumorigenesis and provide insight into the role of this pathway in aRMS.

To assess the role of MST/Hippo signaling in aRMS, MST1/2-floxed (Stk3F/F;Stk4F/F) mice were crossed with an established MSTWT aRMS GEMM driven by conditional expression of Pax3-Foxo1 from the endogenous Pax3 locus and conditional loss of Cdkn2a in Myf6-expressing cells.

Statistical analysis revealed that compared to MSTWT aRMS control animals, MSTNull have significantly accelerated tumorigenesis (median survival 112 vs. 224 days, p< 0.0001) and increased tumor penetrance (76% vs. 27%). MSTNull animals developed tumors disproportionately in the head and neck as compared to control, and incurred multiple tumors per animal. Tumors were analyzed via immunohistochemistry for MyoD and myogenin, markers of RMS, as well as Myf5, an early marker of myogenesis. Tumors in both cohorts were for positive for aRMS markers. Tumor-derived cell lines were used for in vitro cell-based assays and molecular interrogation.

We have identified the MST/Hippo signaling axis as an important tumor suppressor mechanism in aRMS. The rapid onset and increased penetrance of tumorigenesis in this GEMM provides a powerful tool for interrogating aRMS biology and screening novel therapeutics.

Citation Format: Kristianne Oristian, Lisa E. Crose, Rex Bentley, Nina Kuprasertkul, David G. Kirsch, Corinne Linardic. Loss of MST/Hippo signaling promotes tumorigenesis in a genetically engineered mouse model of fusion-positive alveolar rhabdomyosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1939. doi:10.1158/1538-7445.AM2017-1939
Abstract 1956: ABCE1 regulates the translational profile of neuroblastoma to drive tumor progression
Authors: Gao Jixuan , Somers Klaartje , Hannan Katherine M. , Fletcher Jamie I. , Liu Bing , Hannan Ross D. , Pearson Richard B. , Haber Michelle , Norris Murray D. , Henderson Michelle J.
Published: July 2017

Neuroblastoma is the most common extracranial solid tumor in children. A key driver of high-risk neuroblastoma is the MYCN transcription factor which fuels neuroblastoma progression by enhancing metabolic processes including protein translation (1). ABCE1, a member of the ATP-binding cassette (ABC) superfamily of transporters, is a translation factor directly up-regulated by MYCN (2). It is thought to dissociate the 80S ribosome into free 40S and 60S subunits so they can re-initiate translation and provide the protein building blocks required for rapid cell growth and migration (3). High ABCE1 expression is associated with poor clinical outcome in neuroblastoma patients (2). Therefore, we hypothesized that ABCE1 suppression may inhibit the aggressiveness of neuroblastoma by disabling protein synthesis. To test this, we suppressed ABCE1 using siRNAs in MYCN-amplified neuroblastoma cell lines, SK-N-BE(2) and CHP-134, which resulted in severely impaired cell migration (P<0.001) and proliferation (P<0.005). In mice xenografted with SK-N-BE(2) cells, ABCE1 suppression delayed tumor growth (P=0.001) and metastasis (P<0.001). Mechanistically, when polyribosome analysis was used to monitor the impact of ABCE1 expression on protein translation in SK-N-BE(2) cells, loss of ABCE1 reduced the proportion of actively translating ribosomes (P<0.001), leading to lower global protein synthesis (P=0.018). This is the first report of ABCE1 acting as pro-tumorigenic factor in neuroblastoma. Our data imply that targeting the translational machinery through ABCE1 may be an effective therapeutic approach for the treatment of MYC-driven cancers.

1.

Boon, K., Caron, H. N., van Asperen, R., Valentijn, L., Hermus, M. C., van Sluis, P., Roobeek, I., Weis, I., Voûte, P.A., Schwab, M. & Versteeg, R. (2001) EMBO J. 20, 1383-1393.

2.

Porro, A., Haber, M., Diolaiti, D., Iraci, N., Henderson, M., Gherardi, S., Valli, E., Munoz, M.A., Xue, C., Flemming, C., Schwab, M., Wong, J.H., Marshall, G.M., Della Valle, G., Norris, M.D. & Perini, G. (2010) J. Biol. Chem. 285, 19532-19543.

3.

Pisarev, A.V., Skabkin, M.A., Pisareva, V.P., Skabkina, O.V., Rakotondrafara, A.M., Hentze, M.W., Hellen C.U. & Pestova, T.V. (2010). Mol. Cell, 37, 196-210.

Citation Format: Jixuan Gao, Klaartje Somers, Katherine M. Hannan, Jamie I. Fletcher, Bing Liu, Ross D. Hannan, Richard B. Pearson, Michelle Haber, Murray D. Norris, Michelle J. Henderson. ABCE1 regulates the translational profile of neuroblastoma to drive tumor progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1956. doi:10.1158/1538-7445.AM2017-1956
Abstract 196: Identification of small molecule inhibitors of the notch transcriptional activation complex
Authors: Astudillo Luisana , Capobianco Anthony
Published: July 2017

Deregulation of the Notch pathway underlies many aspects of cancer physiology depending on cell type and context. In many human cancers, aberrant Notch activity has been demonstrated to play a role in the initiation and maintenance of the neoplastic phenotype. Notch also plays a central role in cancer stem cells, which may underlie a role in metastasis and resistance to therapy. The important and diverse role played by Notch in cancer makes it an exceedingly attractive target for anti-neoplastic therapeutics. However, the full range of potential targets in the pathway have been under-explored. Through computer-aided drug design we explored potential ligand binding sites and screened for compounds that could disrupt the assembly of the Notch transcriptional activation complex. An in vitro assay that quantitatively measures the assembly of the Notch transcriptional complex on DNA was used for screening. We have recently reported the identification and characterization of a small molecule inhibitor of the Notch transcriptional activation complex, termed Inhibitor of Mastermind Recruitment-1 (IMR-1). Herein we report the characterization of small molecule inhibitors of Notch derived from IMR-1 that were identified through a combination of structure-activity relationship studies and molecular docking simulations. We demonstrate that these compounds inhibit the growth of Notch dependent cell lines and decrease Notch target gene transcription.

Citation Format: Luisana Astudillo, Anthony Capobianco. Identification of small molecule inhibitors of the notch transcriptional activation complex [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 196. doi:10.1158/1538-7445.AM2017-196
Abstract 1962: Regulation of invasion by lysine demethylase 5B in non-small cell lung cancer cells
Authors: Zoeller Elizabeth L. , Konen Jessica , Bell Joshua , Summerbell Emily , Kowalski Jeanne , Marcus Adam , Vertino Paula
Published: July 2017

Histone modifying enzymes are often dysregulated during carcinogenesis and are major contributors to the development of oncogenic features, including proliferation, drug resistance, and metastasis. Given these roles, histone modifiers are promising new targets for oncological therapeutics. One of the enzyme families at the center of this area of research is the lysine demethylase family KDM5, for which several inhibitors are in development. KDM5A and KDM5B are frequently overexpressed or mutated in human non-small cell lung cancer (NSCLC). In overexpression studies, KDM5B promotes invasion and migration of NSCLC cells, whereas invasion and migration of NSCLC cells were decreased following knockdown of KDM5B. Furthermore, in patients with NSCLC, KDM5B is expressed at higher levels in brain metastasis sites when compared to both normal tissues and primary tumors. These findings suggest a role for KDM5B in lung cancer and metastatic spread. However, the precise role of KDM5 family members in lung cancer invasion and metastasis is not known. Recently, we discovered that KDM5B is differentially expressed in cell subtypes within a 3D model of NSCLC collective cell invasion. In this model isolated single cells at the forefront of invasive branches (leader cells) express more 2-4 fold more KDM5B protein than the cells following (follower cells), while KDM5A and KDM5C are evenly expressed across both cell types. KDM5B mRNA expression is similar across cell types suggesting that the differential protein expression is mediated at the posttranscriptional level. Interestingly, global H3K4me3 levels are decreased in leader cells as compared to follower cells, supporting the idea that the lysine demethylases targeting this residue may be expressed at higher levels in leader cells. Collectively, these data suggest that KDM5B could be contributing to initiation of invasion at the primary site and thus, promoting metastasis. Given the rising prominence of therapeutic inhibitors of KDM5 family members, a better understanding how KDM5B contributes to cell invasion may lead to a new approach to the prevention of metastasis.

Citation Format: Elizabeth L. Zoeller, Jessica Konen, Joshua Bell, Emily Summerbell, Jeanne Kowalski, Adam Marcus, Paula Vertino. Regulation of invasion by lysine demethylase 5B in non-small cell lung cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1962. doi:10.1158/1538-7445.AM2017-1962
Abstract 1964: Identification of a novel molecule target for the detection, prediction, and treatment of peritoneal metastasis of gastric cancer
Authors: Kanda Mitsuro , Tanaka Haruyoshi , Shimizu Dai , Kobayashi Daisuke , Tanaka Chie , Takami Hideki , Hayashi Masamichi , Iwata Naoki , Niwa Yukiko , Yamada Suguru , Fujii Tsutomu , Nakayama Goro , Sugimoto Hiroyuki , Koike Masahiko , Fujiwara Michitaka , Kodera Yasuhiro
Published: July 2017

Background: Advanced gastric cancer (GC) frequently recurs because of undetected micrometastases even when disease is localized and patients undergo curative resection. Moreover, peritoneal metastasis is fatal. We aimed to develop novel diagnostic and therapeutic targets specific for peritoneal metastasis of GC to improve management.

Methods: We conducted a metastatic pathway-specific transcriptome analysis to identify candidate biomarkers comprising 340 patients allocated to discovery and validation sets (1:2) to evaluate the diagnostic and predictive value. The mRNA and protein levels in primary GC tissues were compared with patients’ clinical characteristics and survival. The effects of siRNA-mediated knockdown on phenotype and fluorouracil sensitivity of GC cells were evaluated in vitro, and the therapeutic effects of siRNAs were evaluated using a mouse xenograft model.

Results: Synaptotagmin VIII (SYT8) was identified as a candidate biomarker specific to peritoneal metastasis. SYT8 levels were elevated in the validation set comprising patients with peritoneal recurrence or metastasis. High SYT8 levels were significantly and specifically associated with peritoneal metastasis, and served as an independent prognostic marker for peritoneal recurrence-free survival of patients with stage II/III GC. The survival difference between high and low SYT8 levels was associated with patients who received adjuvant chemotherapy. Inhibition of SYT8 expression by GC cells correlated with decreased invasion, migration, and fluorouracil resistance. Intraperitoneal administration of SYT8-siRNA inhibited the growth of peritoneal nodules and prolonged survival of mice engrafted with GC cells.

Conclusions: SYT8 expression represents a promising diagnostic and predictive biomarker for peritoneal metastasis of GC.

Citation Format: Mitsuro Kanda, Haruyoshi Tanaka, Dai Shimizu, Daisuke Kobayashi, Chie Tanaka, Hideki Takami, Masamichi Hayashi, Naoki Iwata, Yukiko Niwa, Suguru Yamada, Tsutomu Fujii, Goro Nakayama, Hiroyuki Sugimoto, Masahiko Koike, Michitaka Fujiwara, Yasuhiro Kodera. Identification of a novel molecule target for the detection, prediction, and treatment of peritoneal metastasis of gastric cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1964. doi:10.1158/1538-7445.AM2017-1964