Cell Trait Research Articles

Abstract 4138: Immunological profile of metastatic or recurrent breast cancer patients

Abstract Background: We previously reported about immune suppression in breast cancer patients at this meeting. In brief, tumor tissue specimens and peripheral blood mononuclear cells (PBMC) were analyzed in 50 early or advanced breast cancer (BC) patients. To compare between 38 early and 12 advanced BC cases, CD163-positive tumor cells and CCR4-positive tumor cells were detected more frequently in advanced cases than in early cases. Regulatory T (Treg) cells in PBMC significantly increased in percentage of the population in 37 BC patients than in 21 healthy volunteers (AACR 2011). In addition, several cytokines were examined in that cohort and plasma IL-17A had significantly higher levels in early BC than in advanced BC (AACR 2013). Then, we examined their prognosis and immunological profile. Patients and methods: Treg cells were examined by counting CD4+CD25highCD127low/-cells in PBMC with flow cytometry analysis. Immunohistochemical evaluation of tumor specimens was performed with monoclonal antibodies of HLA-ABC and DR, CD56, CD68, CD83, CD163 and CCR4. The number of stained cells was analyzed using a semiquantitative ordinal scale ranging from 0 to 3 (0, +/-, ++, +++). Human IL-2, IL-4, IL-6, IL-10, TNF, INFγ and IL-17A were measured using cytometric beads array system. Most patients received chemotherapy, hormonal therapy, and/or anti-HER2 therapy on the basis of intrinsic subtype and breast irradiation after breast-conserving surgery. Results and discussions: At the median follow-up of 7 years after blood sample collection, only 2 operable patients relapsed and 3 patients including 2 cases of stage IV died of disease. Of 5 cases of stage IV or recurrent BC, CD163 and/or CCR4 were strongly stained positive in tumor cells. There were significant differences of staining intensity in CD163 and CCR4-positive tumor cells between those cases and the rest 45 cases (p<0.01 at Chi-square test). However, other immune cell profiles, Treg cells in PBMC and cytokines in plasma had no trend between them. Several reports demonstrated that cancer patients with CD163-positive tumor-infiltrating macrophages and CD163-positive tumor cells had poor prognosis due to tumor-associated macrophage. Phenotypic macrophage traits in cancer cells, like CD163 expression, may be explained by heterotypic cell fusion between monocytes/macrophages and cancer cells. Conclusion: Targeted therapy against M2 macrophage or CCR4 is considered as a promising strategy of advanced breast cancer. Citation Format: Shigeru Imoto, Takayuki Ueno, Hirotsugu Isaka, Hiroki Ito, Kaisuke Miyamoto, Tomohiro Chiba, Hiroshi Kamma. Immunological profile of metastatic or recurrent breast cancer patients. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4138.

Abstract 1894: Understanding the role of microRNA-206 in breast cancer

Abstract MicroRNAs are short non-coding RNA sequences that that can modify genes in post transcriptional manner and are often de-regulated in cancer. Cancer stem cells, a subset of cancer cells with stem cell properties, are considered the root of tumorigenesis and seeds of metastasis. In this study we are interested in elucidating the role of miR-206 in breast cancer stem cell-mediated tumorigenesis and metastasis. We have observed that microRNA-206 (miR-206) regulate the metastatic traits of breast cancer cells, both in vitro and in vivo. Our studies have elucidated a signaling cascade that culminates in the silencing of cytokine (such as interleukin) and integrin expression. Knockdown of the interleukin and integrin genes by siRNAs mimics the phenotype of miR-206 expression in breast cancer cells. DNA content analysis using propidium iodide staining revealed that knockdown of miR-206 targets as well as over-expression of miR-206 in MDA-MB-231 cells regulates cell cycle arrest. Our results indicate that miR-206 mediates its regulatory effects through inhibiting multiple targets. MiR-206 may serve as a new therapeutic candidate for the future treatment of breast cancer. Citation Format: Valery Adorno-Cruz, Huiping Liu. Understanding the role of microRNA-206 in breast cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1894.

Quality Indicator Development for Positive Screen Follow-up for Sickle Cell Disease and Trait

Extensive variation exists in the follow-up of positive screens for sickle cell disease. Limited quality indicators exist to measure if the public health goals of screening-early initiation of treatment and enrollment to care-are being achieved. This manuscript focuses on the development of quality indicators related to the follow-up care for individuals identified with sickle cell disease and trait through screening processes. The authors used a modified Delphi method to develop the indicators. The process included a comprehensive literature review with rating of the evidence followed by ratings of draft indicators by an expert panel held in September 2012. The expert panel was nominated by leaders of various professional societies, the Health Resources and Services Administration, and the National Heart, Lung, and Blood Institute and met face to face to discuss and rate each indicator. The panel recommended nine quality indicators focused on key aspects of follow-up care for individuals with positive screens for sickle cell disease and trait. Public health programs and healthcare institutions can use these indicators to assess the quality of follow-up care and provide a basis for improvement efforts to ensure appropriate family education, early initiation of treatment, and appropriate referral to care for individuals identified with sickle cell disease and trait.

Silencing FOXP2 in breast cancer cells promotes cancer stem cell traits and metastasis

In a recent article in Cell Stem Cell, we showed that mesenchymal stem cells (MSCs), progenitor cells that populate the breast tumor stroma, induce microRNA-mediated FOXP2 repression in breast cancer cells (BCCs), thus promoting cancer stem cell (CSC) and metastatic traits. Here, we discuss the implications of these findings for understanding metastatic CSC genesis.

Abstract A130: Immune regulation of EMT and metastatic competence in breast tumor progression

Abstract Dynamic interactions between cancer cells and their surrounding microenvironment influence tumor survival, growth and metastasis. The tumor microenvironment harbors a complex variety of cells including lymphocytes and macrophages that produce a host of cytokines, chemokines, growth factors or interleukins, all of which presumably work in coordination to determine net tumor biology. Immune cells can offer a critical check-point in tumor progression. Interestingly however, infiltration of the tumor by immune cells appears to have dual functionality - they can reportedly either be pro-metastatic or anti-metastatic in their contribution. These clearly conflicting roles of immune cells limit our comprehension of their actual input to tumor advancement. The presence of tumor infiltrating lymphocytes has emerged as a good prognostic marker for a variety of cancers. However, the key molecular factors that regulate the cross-talk between tumor cells and lymphocytes, and the underlying signaling pathways are still ill-defined. Moreover, the impact of lymphocytes of the inflammatory tumor microenvironment, on the epithelial-mesenchymal plasticity and associated metastatic traits in breast cancer cells, is incompletely understood. We previously demonstrated that two key EMT/CSC factors - FOXC2 and Twist - are critical requirements for breast carcinoma cells to metastasize. The objective of this study is to systematically investigate the role of immune cells in EMT/CSC-dictated breast tumor progression using two isogenic breast cancer cell lines (67NR and 4T1) that form primary mammary tumors similarly, but differ drastically in their ability to metastasize. Citation Format: Robiya Joseph, Rama Soundararajan, Anurag Paranjape, Sendurai Mani. Immune regulation of EMT and metastatic competence in breast tumor progression. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A130.

Epigenetic Activation of TWIST1 by MTDH Promotes Cancer Stem–like Cell Traits in Breast Cancer

Cancer stem-like cells (CSC) are a cell subpopulation that can reinitiate tumors, resist chemotherapy, and give rise to metastases. Metadherin (MTDH) contributes widely to tumor growth, drug resistance, relapse, and metastasis, but its molecular mechanisms of action are not well understood. Here, we report that MTDH drives CSC expansion by promoting the expression of TWIST1, a transcription factor critical for cancer cell stemness and metastasis. MTDH activates TWIST1 expression indirectly by facilitating histone H3 acetylation on the TWIST1 promoter, a process mediated by the histone acetyltransferase CBP. Mechanistic investigations showed that MTDH interacts with CBP and prevents its ubiquitin-mediated degradation, licensing its transcriptional activation of TWIST1. In clinical specimens of breast cancer, MTDH expression correlates positively with TWIST1 expression and CSC abundance. Overall, our work revealed that MTDH promotes CSC accumulation and breast tumorigenicity by regulating TWIST1, deepening the understanding of MTDH function in cancer.

Abstract 2036: Keratin 17 mediates p27KIP1-nuclear export, proliferative signaling and tumor growth

Abstract One of the most fundamental traits of cancer cells is the ability to sustain proliferation by circumventing cell cycle regulatory programs normally controlled by tumor suppressors. Keratin 17 (K17) is not expressed in most somatic tissues but is overexpressed and is a negative prognostic marker in cervical, breast, ovarian, and gastric carcinomas. The mechanisms by which K17 contributes to cancer-related signaling, however, remain unknown. Here, we report that nuclear-localized K17 interacts with tumor suppressor p27KIP1 and mediates its nuclear export, maintaining proliferation. After validating K17 is a prognostic marker in cervical cancer, independent of tumor stage, we performed in vitro and in vivo experiments to investigate the role of K17 in proliferative signaling and tumor growth using loss- and gain- of function approaches in cervical cancer and other cancer-derived cell lines. We found that K17 expression maintains proliferation, while silencing K17 induces G1 arrest by nuclear accumulation and stabilization of tumor suppressors p27KIP1 and pRb. During G1, K17 localizes in the nucleus, mediated by a classical bipartite nuclear localization signal (NLS) (AA385-400) that was identified by in silico analysis. This NLS is specific among keratins and present only in primates but not in other species. To our knowledge, this is the first report that a keratin has a NLS and is capable of undergoing nuclear translocation. During G1, p27KIP1is exported from the nucleus in a CRM1-dependent manner and is subsequently degraded, triggering G1/S transition. p27KIP1 lacks the classical leucine-rich nuclear export signal (NES) and requires an adaptor for CRM1-exportin binding. After confirming the binding of K17 and p27KIP1 within the nucleus, we performed in silico analyses and identified a leucine-rich NES required for CRM1-binding in K17 (AA194-199), which was further validated by a 3-fold nuclear retention of K17 and p27KIP1 after leptomycin B treatment. We introduced point mutations within the K17-NES (mNES) and the K17-NLS (mNLS). Nuclear p27KIP1 was lost in cells expressing wild-type K17. In contrast, nuclear p27KIP1 levels were 3-fold higher in cells expressing either mNLS or mNES. Furthermore, nuclear localization of K17 was abolished in mNLS cells. Xenografts of cervical cancer cells showed that tumors derived from K17 expressing cells were 3-times larger than those derived from K17 knockdown cells, the latter showing increased nuclear p27KIP1 and decreased PCNA and Ki67 expression. Finally, we identified an inverse correlation between K17 and p27KIP1 expression in human cervical cancer specimens, intertumorally and intratumorally. These data suggest a model in which nuclear-localized K17 acts as an oncoprotein promoting G1/S transition in cancer cells, by mediating exportin binding and nuclear translocation of tumor suppressor p27KIP1, contributing to sustained proliferation, tumor aggressiveness and poor-patient outcome. Citation Format: Luisa F. Escobar-Hoyos, Ruchi Shah, Lucia Roa-Peña, Nilofar Najafian, Elizabeth Vanner, Anna Banach, Erik Nielsen, Ramsey Al-Khalil, Ali Akalin, David Talmage, Kenneth Shroyer. Keratin 17 mediates p27KIP1-nuclear export, proliferative signaling and tumor growth. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2036. doi:10.1158/1538-7445.AM2015-2036

The downregulation of ΔNp63 in p53-deficient mouse epidermal tumors favors metastatic behavior.

The TP63 gene codes for two major isoform types, TAp63 and ΔNp63, with probable opposite roles in tumorigenesis. The ΔNp63α protein is frequently amplified and overexpressed in different epithelial tumors. Accordingly, it has been considered a potential oncogene. Nonetheless, a possible metastatic suppressor activity has also been suggested based on the experimental observation that its expression is reduced or even absent in advanced invasive tumors. Such metastatic suppressor activities are often related to tumors bearing point mutated TP53 gene. However, its potential roles in TP53-deficient tumors are poorly characterized. Here we show that in spontaneous tumors, induced by the epidermal-specific Trp53 ablation, the reduction of ΔNp63 expression is an early event, whereas it is re-expressed in the lung metastatic lesions. Using knock down and ectopic expression approaches, we show that ΔNp63 expression opposes the epithelial-mesenchymal transition and reduces the metastatic potential of the cells. This process occurs through the modulation of ΔNp63-dependent downstream targets (including transcription factors and microRNAs) likely to play metastatic roles. Further, ΔNp63 also favors the expression of factors involved in iPS reprogramming, thus suggesting that it can also modulate specific stem cell traits in mouse epidermal tumor cells. Overall, our data assign antimetastatic roles to ΔNp63 in the context of p53 deficiency and epidermis.

Dose-dependent effects of allopurinol on human foreskin fibroblast cells and human umbilical vein endothelial cells under hypoxia.

Allopurinol, an inhibitor of xanthine oxidase, has been used in clinical trials of patients with cardiovascular and chronic kidney disease. These are two pathologies with extensive links to hypoxia and activation of the transcription factor hypoxia inducible factor (HIF) family. Here we analysed the effects of allopurinol treatment in two different cellular models, and their response to hypoxia. We explored the dose-dependent effect of allopurinol on Human Foreskin Fibroblasts (HFF) and Human Umbilical Vein Endothelial Cells (HUVEC) under hypoxia and normoxia. Under normoxia and hypoxia, high dose allopurinol reduced the accumulation of HIF-1α protein in HFF and HUVEC cells. Allopurinol had only marginal effects on HIF-1α mRNA level in both cellular systems. Interestingly, allopurinol effects over the HIF system were independent of prolyl-hydroxylase activity. Finally, allopurinol treatment reduced angiogenesis traits in HUVEC cells in an in vitro model. Taken together these results indicate that high doses of allopurinol inhibits the HIF system and pro-angiogenic traits in cells.

Possible mechanisms of lack of dentin bridge formation in response to calcium hydroxide in primary teeth

Introduction: The usage of Calcium hydroxide (CaOH 2 ) has wide applications due to the property of osteo-inductive, protective, and antibacterial actions. However, it is not used in primary teeth, as it fails to form reparative dentin and the exact mechanism has not been explained. The hypothesis: The authors propose an explanation that lack of dentin bridge formation in response to (CaOH 2 ) in primary teeth could be multifactorial: inability of the deciduous stem cells to generate complete dentin-pulp-like tissue; the absence of calcium-magnesium-dependent adenosine triphosphatase (Ca-Mg ATPase) in the odontoblasts; the pre-existing predilection of deciduous dentine pulp to form odontoclasts; the solubility of (CaOH 2 ). Evaluation of the hypothesis: The hypothesis discusses the innate traits of the deciduous stem cells that lack the ability to form the dentin bridge, the absence of Ca-Mg ATPase enzyme and increased solubility of (CaOH 2 ) together fail to stimulate the odontoblasts. Alternatively, pre-existing progenitor cells with proclivity to change into odontoclasts may cause internal resorption and hamper formation of reparative dentin.

Abstract 4957: Role of tumor-derived exosomes in growth and metastasis of breast cancer

Abstract Breast cancer (BC) mortality rates have decreased over last 30 years, largely due to early detection and improved treatment modalities, particularly targeted therapies. However, metastasis relentlessly drives mortality. The pervasive mortality from metastasis highlights the shortcomings of traditionally accepted hypotheses on the metastatic process. The mechanisms by which malignant tumors leave the primary tumor site, invade lymphatics, and metastasize to regional lymph nodes are complex and interrelated. Exosomes are biological nanovesicles (∼30-100 nm) of endocytic origin. Exosomes mediate local and horizontal transfer of information, such as mRNAs, miRNAs and proteins. They are indicated to play a significant role in the genesis of tumor, growth and metastasis. Recent advances in the exosome biology suggest that tumor-derived exosomes (TDEs) are critical mediators of tumorigenesis and can incite a pro-angiogenic signaling program capable of remodeling tissue matrices. However, the role of TDEs in BC metastasis remains elusive. In this study, we examined the role of exosomes in breast cancer metastasis using cell culture studies. We made the following observations: i) exosomes derived from BC cells had a mean size of 92 nm and carried exosomal marker proteins CD63 and CD81, ii) uptake of exosomes from highly metastatic BC cells (MDA-MB-231) by non-metastatic recipient cells (BT474 and T47D), iii) faster wound healing rate, increased migratory behavior, and invasiveness in recipient cells treated with metastatic BC exosomes, iv) increased endothelial cell tube formation, reflective of angiogenesis ability with exosomes from metastatic BC cells, v) modulation in expression levels of key EMT-associated proteins in non-metastatic cells treated with metastatic BC exosomes favoring EMT shift, vi) difference in metastasis-associated miRNAs levels in exosomes from invasive and non-invasive BC cells. Our data that suggest exosomes from metastatic BC cells carry the cargo essential for EMT and have the ability to change phenotypic traits of the recipient cells. Determining the role of exosomes in BC metastasis and characterization of BC exosomes components (proteins, mRNA and miRNA) will have great potential to uncover new targets and lead to new therapies for metastatic disease. Citation Format: Radha Munagala, Ramesh Gupta. Role of tumor-derived exosomes in growth and metastasis of breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4957. doi:10.1158/1538-7445.AM2014-4957

Abstract 4872: A DKK1 dependent crosstalk between TGFβ2-snail1 and miRNAs contributes to chemoresistance in mesenchymal-like breast cancer

Abstract The role of TGFβ in chemoresponse and progression of breast cancer (BC) remains unclarified, here we investigated the role of TGFβ2 in BC by taking into account BC heterogeneity. mRNA microarray identified TGFβ2 was upregulated in 5-Fu resistant MCF-7 BC cells (MCF-7/5-Fu) with EMT phenotype. TGFβ2 in mRNA and protein from MCF-7/5-Fu and triple-negative BC (TN-BC) cell lines MDA-MB-231, HCC38 and BT-549 with mesenchymal morphology was higher than luminal BC cell lines MCF-7, T47D, BT-474 and SKBR3. TGFβ signal was more active in mesenchymal BC cells than luminal BC cells charactered by p-smad2, p-smad3 and snial1 upregulated. TGFβ signal inhibition with TGFβ receptor (TGFβR) inhibitor SD208 or TGFβRI targeted shRNA reversed the mesenchymal phenotype accompanied with invasion inhibition, BC stem cell (BCSC) traits measured by CD44+CD24- subpopulation analysis and mammosphere assay, and resistance to 5-Fu and Paclitaxel, but played limited effect on MCF-7/5-Fu cells. As the same, TGFβ2 only partially induced EMT phenotype in MCF-7, T47D, BT474 and SKBR3 cells. miRNA microarray indentified downregulation of miR-141, miR-145 and miR-200a (Tβ2-miRNAs) in mesenchymal BC cells compared to lumial BC cells. Lentivirus system with two pre-miRNA units enhanced Tβ2-miRNAs expression accompanied with TGFβ2 downregulation and reversion of mesenchymal phenotype and drug resistance in mesenchymal BC cells. Luciferase activity assay also strongly suggested Tβ2-miRNAs directly target TGFβ2 synergistically. TGFβ signal inhibition and snail1 knockdown upregulated Tβ2-miRNAs in mesenchymal BC cells. ChIP assay showed snail1 bound to promoters of Tβ2-miRNAs, combined with promoter luciferase assay suggested snail1 transcriptionaly repress Tβ2-miRNAs expression in mesenchymal BC cells. However, DKK1 upregulated Tβ2-miRNAs expression in mesenchymal BC cells, but with limited effect in snail1 knockdown cells, suggesting DKK1 could reverse the repressive effect of snail1 on miRNAs expression. TGFβ2 upregulated Tβ2-miRNAs expression in MCF-7 and T47D cells with normal DKK1 expression, and snail1 konckdown abolished the effect of TGFβ2. In addition, using a tissue microarray of 93 BC biopsies containing 42 TN-BC, we found TGFβ2 and snail1 protein were present at higher levels in TN-BC than in luminal BC, and miRNAs measured by ISH and DKK1 protein were present at lower levels. Furthermore, the result of Tβ2-miRNAs detection in the serum samples from 76 BC patients including 34 TN-BC showed Tβ2-miRNAs abundance in serum samples from TN-BC was much lower. In conclusion, the role of TGFβ in BC is cell type dependent and our findings suggest TGFβ2 protein and Tβ2-miRNAs levels in serum will be valuable prognostic biomarkers and therapeutic strategies for mesenchymal BC. Note: This abstract was not presented at the meeting. Citation Format: Guopei Zheng, Xiaoting Jia, Zijuan Zhang, Zhijie Zhang, Chen Qu, Jiang Yin, Zhimin He. A DKK1 dependent crosstalk between TGFβ2-snail1 and miRNAs contributes to chemoresistance in mesenchymal-like breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4872. doi:10.1158/1538-7445.AM2014-4872

Mesenchymal stem cell therapy for the treatment of amyotrophic lateral sclerosis: signals for hope?

Based on the distinctive cellular, molecular and immunomodulatory traits of mesenchymal stem cells (MSC), it has been postulated that these cells may play a critical role in regenerative medicine. In addition to the participation of MSC in the repair of mesodermal-derived tissues (bone, cartilage), robust data have suggested that MSC may also play a reparative role in conditions involving damage of cells of ectodermal origin. The above content has been supported by the capability of MSC to differentiate into neuron-like cells as well as by a competence to generate a 'neuroprotective' environment. In turn, several preclinical studies have put forward the concept that MSC therapy may represent an option for the treatment of several neurological disorders and injuries, including amyotrophic lateral sclerosis. We expect that the above foundations, which have inspired this review, may result in the founding of an effective and/or palliative therapy for amyotrophic lateral sclerosis.

Genetic drivers of metastatic dissemination in sonic hedgehog medulloblastoma.

Leptomeningeal dissemination (LMD), the metastatic spread of tumor cells via the cerebrospinal fluid to the brain and spinal cord, is an ominous prognostic sign for patients with the pediatric brain tumor medulloblastoma. The need to reduce the risk of LMD has driven the development of aggressive treatment regimens, which cause disabling neurotoxic side effects in long-term survivors. Transposon-mediated mutagenesis studies in mice have revealed numerous candidate metastasis genes. Understanding how these genes drive LMD will require functional assessment using in vivo and cell culture models of medulloblastoma. We analyzed two genes that were sites of frequent transposon insertion and highly expressed in human medulloblastomas: Arnt (aryl hydrocarbon receptor nuclear translocator) and Gdi2 (GDP dissociation inhibitor 2). Here we show that ectopic expression of Arnt and Gdi2 promoted LMD in mice bearing Sonic hedgehog (Shh)-induced medulloblastomas. We overexpressed Arnt and Gdi2 in a human medulloblastoma cell line (DAOY) and an immortalized, nontransformed cell line derived from mouse granule neuron precursors (SHH-NPD) and quantified migration, invasiveness, and anchorage-independent growth, cell traits that are associated with metastatic competence in carcinomas. In SHH-NPD cells. Arnt and Gdi2 stimulated all three traits. In DAOY cells, Arnt had the same effects, but Gdi2 stimulated invasiveness only. These results support a mechanism whereby Arnt and Gdi2 cause cells to detach from the primary tumor mass by increasing cell motility and invasiveness. By conferring to tumor cells the ability to proliferate without surface attachment, Arnt and Gdi2 favor the formation of stable colonies of cells capable of seeding the leptomeninges.Electronic supplementary materialThe online version of this article (doi:10.1186/s40478-014-0085-y) contains supplementary material, which is available to authorized users.

Activation of the germ-cell potential of human bone marrow-derived cells by a chemical carcinogen

Embryonic/germ cell traits are common in malignant tumors and are thought to be involved in malignant tumor behaviors. The reasons why tumors show strong embryonic/germline traits (displaced germ cells or gametogenic programming reactivation) are controversial. Here, we show that a chemical carcinogen, 3-methyl-cholanthrene (3-MCA), can trigger the germ-cell potential of human bone marrow-derived cells (hBMDCs). 3-MCA promoted the generation of germ cell-like cells from induced hBMDCs that had undergone malignant transformation, whereas similar results were not observed in the parallel hBMDC culture at the same time point. The malignant transformed hBMDCs spontaneously and more efficiently generated into germ cell-like cells even at the single-cell level. The germ cell-like cells from induced hBMDCs were similar to natural germ cells in many aspects, including morphology, gene expression, proliferation, migration, further development, and teratocarcinoma formation. Therefore, our results demonstrate that a chemical carcinogen can reactivate the germline phenotypes of human somatic tissue-derived cells, which might provide a novel idea to tumor biology and therapy.

Genetic drivers of metastatic dissemination in sonic hedgehog medulloblastoma

Leptomeningeal dissemination (LMD), the metastatic spread of tumor cells via the cerebrospinal fluid to the brain and spinal cord, is an ominous prognostic sign for patients with the pediatric brain tumor medulloblastoma. The need to reduce the risk of LMD has driven the development of aggressive treatment regimens, which cause disabling neurotoxic side effects in long-term survivors. Transposon-mediated mutagenesis studies in mice have revealed numerous candidate metastasis genes. Understanding how these genes drive LMD will require functional assessment using in vivo and cell culture models of medulloblastoma. We analyzed two genes that were sites of frequent transposon insertion and highly expressed in human medulloblastomas: Arnt (aryl hydrocarbon receptor nuclear translocator) and Gdi2 (GDP dissociation inhibitor 2). Here we show that ectopic expression of Arnt and Gdi2 promoted LMD in mice bearing Sonic hedgehog (Shh)-induced medulloblastomas. We overexpressed Arnt and Gdi2 in a human medulloblastoma cell line (DAOY) and an immortalized, nontransformed cell line derived from mouse granule neuron precursors (SHH-NPD) and quantified migration, invasiveness, and anchorage-independent growth, cell traits that are associated with metastatic competence in carcinomas. In SHH-NPD cells. Arnt and Gdi2 stimulated all three traits. In DAOY cells, Arnt had the same effects, but Gdi2 stimulated invasiveness only. These results support a mechanism whereby Arnt and Gdi2 cause cells to detach from the primary tumor mass by increasing cell motility and invasiveness. By conferring to tumor cells the ability to proliferate without surface attachment, Arnt and Gdi2 favor the formation of stable colonies of cells capable of seeding the leptomeninges.

Wharton’s jelly mesenchymal stem cells differentiation into hepatocyte-like cells: functional characterization and expression of immunomodulatory molecules

Mesenchymal stem cells derived from Wharton’s jelly (WJ-MSCs) recently emerged as promising tools for cellular therapy due to their ability to differentiate into diverse cell types and their immunomodulatory features. Little is known on the expression of immunomodulatory molecules in mature cells differentiated from WJ-MSCs, therefore we aimed to characterize the extent of maintenance of the naive traits of these cells also in a highly specialized differentiated counterpart. WJ-MSCs were differentiated into hepatocyte-like cells (HLCs) with a four weeks protocol. RT-PCR, flow cytometry, IHC and ICC were performed to assess expression of key markers in both undifferentiated and differentiated cells. Hepatocyte specific assays such as PAS staining, CYP3A4 induction and activity, G6Pase activity, were performed to demonstrate the acquisition of traits of the mature hepatocyte phenotype. WJ-MSCs were successfully differentiated into HLCs using a multi-step protocol. These cells were able to store glycogen, incorporate specific live cells stains, perform enzymatic reactions and metabolic activities which are usually featured by mature hepatocytes. In addition, WJ-MSCs did express several immune-related molecules, for which an immunomodulatory role has been demonstrated both in vitro and in vivo. We demonstrated for the first time that key molecules as HLA-E and B7-H3 are expressed also by HLCs derived from WJ-MSCs. Our data showed for the first time that HLCs mostly maintain the expression of immune-modulating molecules, together with new markers and functional features of mature cells. In multiple pathologic conditions the intrinsic immunomodulatory ability of differentiated cells may help survive the interaction with the host immune system, even in the absence of a specific immunosuppressive therapy. This, together with the acquisition of key mature hepatocyte functions, may render WJ-MSCs promising in cell therapy applications for liver diseases.

High expression of leptin receptor leads to temozolomide resistance with exhibiting stem/progenitor cell features in gliobalastoma

Glioblastoma is a highly aggressive malignant disease with notable resistance to chemotherapy. In this study, we found that leptin receptor (ObR)-positive glioblastoma cells were resistant to temozolomide (TMZ), and TMZ-resistant cells exhibited high expression of ObR. ObR can serve as a marker to enrich glioblastoma cells with some stem/progenitor cell traits, which explained the reason for TMZ resistance of ObR+ cells. STAT3-mediated SOX2/OCT4 signaling axis maintained the stem/progenitor cell properties of ObR+ cells, which indirectly regulated glioblastoma TMZ resistance. These findings gain insight into the molecular link between obesity and glioblastoma, and better understanding of this drug-resistant population may lead to the development of more effective therapeutic interventions for glioblastoma.

Abstract A100: KLF8 induces breast stemness and chemoresistance via miRNAs associated with EMT.

Abstract The mechanisms behind the induction of stem cell properties by the epithelial to mesenchymal transition (EMT) remain largely undefined. Here we report the identification of the microRNA-146a (miR-146a) as a common target of the known EMT-inducers Krüppel-like factor 8 (KLF8) and TGF-β. Upon KLF8 overexpression or TGF-β treatment, a significant portion of the MCF-10A cells gained stem cell traits as demonstrated by an increase in the expression of CD44high/CD24low, the activity of aldehyde dehydrogenase (ALDH) and the mammosphere forming capability. Along with this change, the expression of miR-146a was highly upregulated in the cells. Consistently, we found that miR-146a was aberrantly co-overexpressed with KLF8 in a panel of invasive human breast cancer cell lines. Ectopic expression of KLF8 failed to induce the stem cell traits in the MCF-10A cells if the cells were pre-treated with miR-146a inhibitor, whereas overexpression of miR-146a in the MCF-10A cells alone was sufficient to induce the stem cell traits. Importantly, this induction of miR-146a-dependent stem traits by KLF8 was well correlated to the cell resistance to the cytotoxic effects of chemotherapeutic drugs such as paclitaxel. Co-staining and luciferase reporter analyses indicated that miR-146a targets the 3’-UTR of the Notch signaling inhibitor NUMB for translational inhibition. Overexpression of KLF8 dramatically potentiated the tumorigenecity of MCF-10A cells expressing the H-Ras oncogene, which was accompanied by a loss of NUMB expression in the tumors. Taken together, this study identifies a novel role and mechanism for KLF8 in inducing pro-tumorigenic mammary stemness and chemoresistance via miR-146a potentially by activating Notch signaling. This mechanism could be exploited as a therapeutic target against drug resisting breast cancer. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A100. Citation Format: Xianhui Wang, Heng Lu, Tianshu Li, Lin Yu, Gang Liu, Xu Peng, Jihe Zhao. KLF8 induces breast stemness and chemoresistance via miRNAs associated with EMT. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A100.

Abstract A160: Characterization of novel regulators for epithelial-mesenchymal transition.

Abstract The epithelial-mesenchymal transition (EMT) confers mesenchymal properties on epithelial cells and has been closely associated with the acquisition of aggressive traits of breast cancer cells. Recently, a few kinases, such as FYN and c-Met, were reported to participate in regulating EMT. However, the roles of the majority of >700 human kinases in EMT and breast cancer progression are still unknown. Here we carried out human kinase cDNA screens to identify novel regulators of EMT. Many unknown EMT regulators and a few known ones such as PI3K, PDGFRA, FYN and c-Met, were found to up-regulate mesenchymal marker vimentin. Some also increase expression of fibronectin and N-cadherin, as well as EMT transcriptional regulators, such as ZEB1/2, Snail and Slug. At the same time, the cells acquired mesenchymal phenotypes, such as increased migration/invasion activity, enhanced capability to survive and proliferate in growth factor deprivation condition or under drug treatments. Interestingly, FACS analysis also showed a substantial increase in CD44hiCD24low population in kinase-transducted mammary gland epithelial cells. The increase of CD44hi population could be attributed to increase of a CD44 mesenchymal variant - the standard form CD44s and decrease of the epithelial variant CD44 v8-10. And this alternation was associated with down-regulation of ESRP1 and ESRP2, two splicing factors that regulate CD44 splicing and EMT. Furthermore, the mammary gland epithelial cells undergoing EMT showed increase capability of forming mammosphere and differentiating into other lineages, such as osteoblast and adipocyte. Importantly, some of these novel EMT regulators are associated with TNBC, stemness and poor prognostics of human breast cancers. One novel regulator, a poorly studied serine/threonine protein kinase, was tested in vivo and found to be capable of generating primary tumor and metastasis at much higher efficiency. Further study of these novel EMT regulators promises to provide new mechanisms of breast cancer progression, and may also lead to development of new prognostic markers and therapeutics. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A160. Citation Format: Linna Li, Emily Lu, Jeffrey Chang, Wenliang Li. Characterization of novel regulators for epithelial-mesenchymal transition. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A160.