Abstract: Cancer pathogenesis is restricted by stresses that compromise cell division and survival. In this study, we identify miR-708, a little studied member of a set of microRNAs that have been implicated in stress control, as an important tumor suppressor in renal cell carcinoma (RCC). miR-708 expression was attenuated widely in human RCC specimens. Restoration of miR-708 expression in RCC cell lines decreased cell growth, clonability, invasion, and migration and elicited a dramatic increase in apoptosis. Moreover, intratumoral delivery of miR-708 was sufficient to trigger in vivo regression of established tumors in murine xenograft models of human RCC. Investigation of the targets of miR-708 identified the inhibitor of apoptosis protein survivin as important. siRNA-mediated knockdown of survivin partially phenocopied miR-708 overexpression suggesting that the proapoptotic role of miR-708 may be mediated primarily through survivin regulation. Additionally, we identified the E-cadherin regulators ZEB2 and BMI1 as likely miR-708 targets. Taken together, our findings define a major tumor suppressive role for miR-708, which may offer an attractive new target for prognostic and therapeutic intervention in RCC.

Abstract: There is now compelling evidence that gliomas harbor a small population of cells, termed glioma-initiating cells (GICs), characterized by their ability to undergo self-renewal and initiate tumorigenesis. The development of therapeutic strategies targeted toward GIC signaling may improve the treatment of malignant gliomas. The characterization of GICs provides a clue to elucidating histological heterogeneity and treatment failure. The role of the stem cell marker CD133 in the initiation and progression of brain tumors is still uncertain. Here, we review some of the signaling mechanisms involved in GIC biology, such as phosphatase and tensin homolog (PTEN), sonic hedgehog, Notch, and WNT signaling pathways, maternal embryonic leucine-zipper kinase (MELK), BMI1, and Janus kinase signal transducer and activator of transcription (JAK-STAT) signaling. In addition, we discuss the role of microRNAs in GICs by focusing on microRNA-21 regulation by type I interferon.

Abstract: The transcription factor signal transducer and activator of transcription 3 (STAT3) is frequently activated in human cancers. Interestingly, STAT3 also maintains the pluripotency and self-renewal of murine embryonic stem cells, and several tissue stem cell types. To investigate whether STAT3 also maintains the small-intestine crypt stem cell, we conditionally inactivated a Floxed Stat3 allele (Stat3fl) in murine small-intestine crypt stem cells. Following Cre recombinase expression, apoptosis increased in Stat3fl/ experimental crypts relative to Stat3wt/ controls before declining. Control Stat3wt/ mice carrying a Flox-STOP LacZ reporter transgene stably expressed LacZ after Cre induction. In contrast, Stat3fl/intestine LacZ expression initially increased modestly, before declining to background levels. Quantitative PCRs revealed a similar transient in recombined Stat3fl allele levels. Long-term bromodeoxyuridine labelling directly demonstrated that functional STAT3 is required for þ4 to þ6 region label-retaining small-intestine stem cell survival. Rapid clearance of recombined Stat3fl/ cells involves apoptosis potentially induced by elevated c-Myc in non-recombined cells and involves elevated p53 expression and caspase 3 activation. Intriguingly, Stat3fl/ intestine recombination triggered dramatically upregulated polycomb transcriptional repressor Bmi1 – potentially accelerating recombined crypt repopulation. In summary, STAT3 activity is absolutely required for small-intestine crypt stem cell survival at both the þ4 to þ6 label-retaining and crypt base columnar cell locations.

Abstract: Polycomb group (PcG) proteins have recently been shown related to cancer development. The PcG protein EZH2 is involved in progression of prostate and breast can- cers, and has been identified as a molecular marker in breast cancer. Nevertheless, the molecular mechanism by which PcG proteins regulate cancer progression and malignant metastasis is still unclear. PcG proteins methylate H3K27 in undiffer- entiated epithelial cells, resulting in the repression of differ- entiation genes such as HOX. FOXC1 is a member of the Forkhead box transcription factor family, which plays an important role in differentiation, and is involved in eye development. We discovered in this study that the expression of FOXC1 gene was negatively correlated to that of PcG genes, i.e., Bmi1, EZH2, and SUZ12, in MCF-7 and MDA- MB-231 cells. To investigate the regulatory effects of PcG proteins on FOXC1 gene, the two cell lines were transfected with either expression plasmids or siRNA plasmids of Bmi1, EZH2, and SUZ12, and we found that PcGs, especially EZH2, could repress the transcription of FOXC1 gene. Chromatin immunoprecipitation (ChIP) assay showed that histone methylation and acetylation modifications played critical roles in this regulatory process. When FOXC1 was stably transfected into MDA-MB-231 cells, the migration and invasion of the cells were repressed. Moreover, the tumori- genicity and the spontaneous metastatic capability regulated by FOXC1 were determined by using an orthotropic xenograft tumor model of athymic mice with the FOXC1-MDA-MB- 231HM and the GFP-MDA-MB-231HM cells, and the results showed that FOXC1 in MDA-MB-231HM cells inhibited migration and invasion in vitro and reduced the pulmonary metastasis in vivo. Data presented in this report contribute to the understanding of the mechanisms by which EZH2 par- ticipates in tumor development. Keywords FOXC1 � PolycombEZH2 � Metastasis � Breast cancer Abbreviations

Abstract: Background In laryngeal squamous cell carcinoma (SCC), CD133+ cells were found to display cancer stem cell (CSC) characteristics. BMI1 is an oncogene that plays key roles in proliferation in CSCs. However, no published reports have examined the role of BMI1 in laryngeal CSCs. Methods Immunofluorescence staining confirmed the coexpression of BMI1 and CD133. After sorting, real-time polymerase chain reaction (PCR) revealed BMI1 was differentially expressed in CD133+ cells. BMI1 was knocked down and proliferation, colony formation, and apoptosis assays were performed. The influence on CD133+ cells was determined by flow cytometry, sphere-formation assay, and quantitative PCR. Tumorigenicity assays were performed, and the impact on related genes was evaluated. Results BMI1 was highly enriched in CD133+ cells. BMI1 maintained CD133+ cell proliferation and prevented apoptosis. Gene expression analysis suggested BMI1 regulated alternate cellular pathways. Conclusion BMI1 was required to maintain the proliferative capacity of laryngeal CSCs, which may be a molecular target to cure patients with laryngeal SCC. © 2010 Wiley Periodicals, Inc. Head Neck, 2011

Abstract: Cancer stemness is a concept used to describe a minor population of cells (cancer stem cells-CSCs) residing in a tumor, which possess self-renewal properties and are resistant to chemo/radiation therapy. Epithelial-mesenchymal transition (EMT), a major mechanism of cancer metastasis, is a process which generates cells with stem-like properties. The relationship between cancer stemness and EMT is well documented but without detailed mechanistic explanation. Bmi1 belongs to the polycomb repressive complex 1 (PRC1) which maintains self-renewal and stemness. Recent results showed that Twist1, an EMT regulator, directly activates Bmi1 and these two molecules function together to mediate cancer stemness and EMT. These results provide a molecular explanation of the relationship between cancer stemness and EMT. Bmi1 is frequently overexpressed in various types of human cancers and can confer drug resistance. Twist1 is also overexpressed in various human cancers with prognostic significance. The functional interdependence between Twist1 and Bmi1 provides a fresh insight into the molecular mechanism of EMT-induced cancer stemness. Further investigation of the mechanisms mediating EMT and cancer stemness will be helpful in the management and treatment of metastatic cancers.

Abstract: Chronic myeloid leukemia (CML) is a clonal myeloproliferative disorder that is characterized by the presence of a fusion oncogene, BCR-ABL, which encodes a protein with constitutive tyrosine kinase activity. This activity causes excessive production of myeloid cells and their premature release into the circulation. The discovery of tyrosine kinase inhibitors marked a major advance in CML therapy, but these drugs cannot eradicate the disease because they are unable to kill the most primitive, quiescent leukemic stem cells. This review discusses current research in CML and attractive targets that have emerged with potential for eradicating the disease. Several new targets have recently been investigated as potential modulators in myeloid leukemia pathogenesis, including the multiple gene regulators miRNAs, the apparently leukemia-specific cell surface marker IL1RAP, transcription factors such as BMI1 and FOXOs, the tumor suppressors PML and PP2A, and the tyrosine kinase JAK2.

Abstract: Introduction Medulloblastoma is the most frequent type of embryonal tumor in the pediatric population, accounting for 20–25% of all brain tumors in children. Recently, the suspected contribution of the Polycomb group (PcG) genes in medulloblastoma development was described. PcG genes play an important role in developmental processes; they are also involved in the self-renewal of hematopoietic and neural stem cells as well as in malignant transformation.

Abstract: 866 Cell Cycle Volume 10 Issue 6 Skin homeostasis relies on epidermal stem cells (ESC) that are mobilized from their niche located in the basal layer of the interfollicular epithelium (IFE) and the bulge of hair follicles (HF) to fuel highly proliferative transit amplifying compartments (TAC). Differentiation programs then generate the spinous, granular and cornified layers of the IFE or the different lineages of the mature HF. ESC maintenance is orchestrated by a complex signalling network that remains incompletely characterized, including p63-, BMP-, TGFb-, Wnt/b-catenin-, Rac1and Notch-initiated signalling cascades. A recent work by Lacroix, Caramel et al. reveals that E4F1 and the Bmi1-Ink4a/ Arfp53 axis define a novel layer of regulation controlling ESC-dependent skin homeostasis (Fig. 1). E4F1 is an ubiquitously expressed multifunctional protein, originally identified as a transcription factor targeted by the adenoviral oncoprotein E1A. E4F1 also acts as an atypical ubiquitin E3-ligase for p53, and interacts with several components of the p53 pathway, including p14 and the polycomb member Bmi1. We recently generated E4F1 conditional knock-out (KO) mice to circumvent the early embryonic developmental failure of E4F1 KO embryos and address E4F1 functions in adult tissues. E4F1 inactivation in adult skin first induces a transient hyperplasia of the epidermis associated with increased proliferation of keratinocytes with basal/TAC properties, followed by severe ulcerative lesions, broad disorganisation of the IFE, massive hyperkeratosis and ultimately, a permanent loss E4F1 connects the Bmi1-ARF-p53 pathway to epidermal stem cell-dependent skin homeostasis

Abstract: Mainly disclosed is a technique for artificially constructing a cancer stem cell from a cancer cell. A cancer cell is derived into a cancer stem cell having a self-renewal ability and acquiring an undifferentiation potency by introducing, into the cancer cell, (1) a KLF family gene, (2) an OCT family gene, (3) an SOX family gene, and (4) a gene of at least one kind of an undifferentiation-inducing factor selected from the group consisting of NANOG, LIN28, KRAS, BCL2, BMI1 and ERAS, and/or an expression inhibitor of at least one kind of a cancer suppressor gene selected from the group consisting of CDKN2A, FHIT, TP53, RB1, PTEN and LZTS1.

Abstract: Despite the fact that DNA methyltransferases (DNMTs) are pivotal genes for survival of many somatic types and cancer, little is known about the role of DNMTs in the regulation of stem cells and especially stem cell aging. Unlike differentiated cells, stem cells could maintain their self-renewal ability and tolerate hypomethylation in the absence of DNMTs with highly expressed Polycomb (PcG) repressive complex 1 and 2 subunits. Aging is accompanied by a decline in the homeostatic and regenerative capacity of somatic tissues, and stem cells are not exceptional. In adult stem cells, aging causes functional changes in the fate of stem cell progeny; however, less is known about how epigenetic regulation is involved and affects stem cell aging. We recently reported that human umbilical cord blood-derived stem cells (hUCB-SCs), expression of DNMT1 and DNMT3b were decreased during hUCB-SCs are losing their self-renewal and multipotency; moreover, inhibition of DNMTs induced cellular senescence of hUCB-SCs through the downregulation of EZH2 and BMI1 PcG proteins, while delaying the G1/S transition

Abstract: Side population (SP) cell analysis has been used to identify and isolate a minor population of cells with stem cell properties in normal tissues and in many cancers including breast cancer cells. However, the molecular mechanisms that operate in tumor-initiating cells (TICs) in SP fraction remain unclear. The Polycomb group genes, including Bmi1 and Mel-18, have been implicated in the maintenance of hematopoietic stem cells (HSCs) and suggested to be oncogenic and tumor suppressive, respectively, in breast cancer. In this study, we determined the critical role of Mel-18 in the enrichment mechanisms of TICs with the SP phenotype in a mouse breast cancer cell line, MMK3, that was established from a breast cancer developed spontaneously in Mel-18+/- mice. The Mel-18 protein expression level significantly correlates to the percentage of SP fraction in the mouse breast cancer cell line MMK3 series. The comparison between MMK3V3 (V3) cells containing one copy of the Mel-18 gene and MMK3S2 (S2) cells having twice the amount of Mel-18 expression clearly demonstrates the above relationship. Similar results obtained with the percentage of ALDH+ cells in V3 and S2 further confirmed the correlation between protein expression level of Mel-18 and the TICs. More importantly, transplantation of SP and non-SP cells of V3 and S2 cells into the NOD/SCID mice clearly showed that the heterozygous level of Mel-18 leads to the disappearance of enrichment of TICs into SP fraction in vivo. Stem cell pathway focused gene expression profiling of V3 and S2 cells revealed that the genes Abcg2, Aldh1a1 and Dhh were highly down-regulated in V3 compared to S2. These results indicate that the precise Mel-18 expression level controls TIC enrichment mechanisms through the regulation of channel molecule of Abcg2 and functional TIC marker of Aldhlal. In conclusion, our findings revealed the significance of fine-tuning mechanisms for Mel-18 protein expression level in the maintenance of TIC into SP fractions in mouse breast cancer.

Abstract: Glioblastoma Multiforme is a WHO grade IV brain tumor with glial characteristics leading to more years of life lost than any other cancer. A better understanding of GBM biology and valid animal models are the two key elements to improved therapeutic results. We found Deltex1, which is part of an alternative Notch pathway, to activate both the PI3K/PKB and the MAPK/ERK pathway and to induce anti-apoptotic Mcl-1. DTX1 over-expression resulted in increased clonogenic and growth potential and also induced cell migration and invasion. Microarray gene expression analysis identified a DTX1-specific transcriptional program related to the changes in phenotype observed. Patients with low DTX1 levels have a favorable prognosis. Therefore, we propose the alternative Notch pathway via DTX1 as an oncogenic factor in glioblastoma. This could partially explain previous findings linking Notch status to prognosis wherein high Notch2 levels correlated with reduced patient survival. We found activated Notch2 in neural stem cells to induce glioma-inducing-cell features including increased proliferation, reduced apoptosis and astrocytic lineage commitment. These observations were found both in vitro and in vivo using a conditional mouse model. Together, these findings indicate an important role for Notch signaling (both canonical and non-canocical) in high grade glioma, offering a potential treatment target. We have also established an in vivo model of GBM which allowed us to analyze the efficacy of novel treatment regimens. Short term treatment of orthotopic xenograft gliomas in nude mice with histone deacetylase inhibitors and 2-deoxy-D-glucose resulted in prolonged survival, reduced tumor growth and induction of cancer cell apoptosis. Therefore, epigenetic reprogramming in combination with energy deprivation was found to have anti-tumor activity in vivo. In an independent project we identified GSK3β as a downstream target of Bmi1. GSK3β maintains a more stem cell like characteristic in GBM cells, potentially also in the glioma inducing cells. Several inhibitors to GSK3β exist and LiCl, which is often used in the clinic, correlates with reduced glioma incidence. Altogether, we believe we have added considerable knowledge on the biology of gliomas thereby helping to characterize the underlying events of gliomagenesis. Furthermore, we have established proof of principle for a novel treatment strategy in vivo.

Abstract: Review on BMI1 (BMI1 polycomb ring finger oncogene), with data on DNA, on the protein encoded, and where the gene is implicated.

Abstract: Objective To investigate the Bmil protein level in human colorectal cancer specimen and associated clinicopathologieal parameters, and to determine the influence of Bmil on the proliferation and apoptosis of colorectal cancer cells. Methods Bmil protein level was assessed in 85 patients with colorectal cancer and adjacent normal tissue by immunohistochemistry. SW480 cells were transfected with Bmi siRNA plasmid. MTT assay and flow cytometry were used to measure the proliferation and apoptosis of SW480 cells. The expression of Bmil and Bcl-2 were measured by Western blot. Results The positive rate of Broil expression in colorectal cancer tissues was 56.5% （48/85）, significantly higher than that in the adjacent noncancerous tissues [17.6% （15/85）, P〈 0.05） ]. It was found that the overexpression of Bmil was associated with degree of differentiation, status of lymph nodes metastasis, and TNM staging in colorectal cancer （P〈0.05）. After transfection of SW480 with Bmil siRNA, the cell proliferation was inhibited and the apoptosis was significant. The cell proliferation inhibitory rates were 13.1%, 16.5%, and 18.3% at 24 h, 48 h and 72 h after transfection. The apoptotic rates were 15.7%, 45.6%, 40.2%, respectively. Expression of Bmil was downregulated after 48 h, as was that of Bcl-2. Conclusions Bmil expression is associated with the clinicopathological characteristics of colorectal cancer. Blockade of Bmil can inhibit the proliferation and accelerate the apoptosis of colorectal cancer cells. Key words: Colorectal neoplasms; Polycomb,Bmil; RNA interfere

Abstract: Many adult tissue stem cells, such as the cells of the hematopoietic system, gastrointestinal epithelium, brain, epidermis, mammary gland and lung have now been identified, all of them fulfilling a crucial role in supplying organisms with mature cells during normal homeostasis as well as in times of tissue gen­ eration or repair. Two unique features characterize adult stem cells: the abil­ ity to generate new pluripotent stem cells (to self-renew) and the ability to give rise to differentiated progeny that has lost its self-renewal capacity. Our understanding of the mechanisms that determine whether, where and when a stem cell will self-renew or differentiate is still limited, but recent advances have indicated that the stem cell microenvironment, or niche, provides es­ sential cues that direct these cell fate decisions. Moreover, loss of control over these cell fate decisions might lead to cellular transformation and cancer. This review addresses the current understanding of the molecular mechanisms that regulate hematopoietic stem cell self-renewal in the niche, and how leu­ kemic transformation might change the dependency of leukemic stem cells on their microenvironment for self-renewal and survival.

Abstract: Glioblastoma multiforme (GBM) is the most common and lethal primary brain tumor found in adult Even with all the advances made in the field of cancer therapy for the last decade, the prognosis has not significantly changed, with a median survival of less than 1 year Therefore, a better understanding of GBM biology is needed Unraveling the basic mechanisms responsible for the initiation and progression of this tumor may open new fields for the development of efficient therapeutic strategies Proteins of the polycomb (PcG) group family generally operate as transcription repressors and BMI1 is one of the best-characterized member The implication of BMI1 in normal and cancerous stem cells survival, self-renewal and maintenance has been widely investigated In most GBM, a relatively rare cell population, characterized as the tumor initiating cell population, expresses the stem cell marker CD133 In experimental systems, CD133-positive GBM cells are responsible for tumor initiation, maintenance, progression and resistance to chemo/radiotherapy CD133-positive GBM “stem cells” thus possibly represent a valuable and specific cellular target to eradicate the tumor Here, we report on the implication of BMI1 in GBM stem cell self-renewal and how BMI1 is indispensable for GBM tumor establishment and progression in a xenograft mouse model

Abstract: BMI1 is a key component of multiprotein Polycomb repression complex 1 (PRC1), and its disruption in mice induces severe aplastic anemia by early adulthood. The contributing mechanisms responsible for this phenotype remain elusive. Here we show that transformed human cell lines as well as primitive hematopoietic cells exhibit a high frequency of spontaneous chromosome breaks upon BMI1 depletion and are hypersensitive to genotoxic agents. Consistent with these observations, we found that BMI1 is recruited rapidly to DNA damage foci where it blocks transcriptional elongation. We also show that BMI1 contributes to homologous recombination DNA repair and is required for checkpoint recovery. Taken together, our results suggest that BMI1 is critical for the maintenance of chromosome integrity in both normal and transformed cells.

Abstract: Polycomb group protein (PcG)-mediated gene silencing is emerging as an essential developmental regulatory mechanism in eukaryotic organisms. PcGs inactivate or maintain the silenced state of their target chromatin by forming complexes, including Polycomb Repressive Complex 1 (PRC1) and 2 (PRC2). Three PRC2 complexes have been identified and characterized in Arabidopsis; of these, the EMF and VRN complexes suppress flowering by catalyzing the trimethylation of lysine 27 on histone H3 of FLOWER LOCUS T (FT) and FLOWER LOCUS C (FLC). However, little is known about the role of PRC1 in regulating the floral transition, although AtRING1A, AtRING1B, AtBMI1A, and AtBMI1B are believed to regulate shoot apical meristem and embryonic development as components of PRC1. Moreover, among the five RING finger PcGs in the Arabidopsis genome, four have been characterized. Here, we report that the fifth, AtBMI1C, is a novel, ubiquitously expressed nuclear PcG protein and part of PRC1, which is evolutionarily conserved with Psc and BMI1. Overexpression of AtBMI1C caused increased H2A monoubiquitination and flowering defects in Arabidopsis. Both the suppression of FLC and activation of FT were observed in AtBMI1C-overexpressing lines, resulting in early flowering. No change in the H3K27me3 level in FLC chromatin was detected in an AtBMI1C-overexpressing line. Our results suggest that AtBMI1C participates in flowering time control by regulating the expression of FLC; moreover, the repression of FLC by AtBMI1C is not due to the activity of PRC2. Instead, it is likely the result of PRC1 activity, into which AtBMI1C is integrated.

Abstract: Mortality from lung cancer is important worldwide. Recently, epigenetic aberration of lung cancer, not only genomic DNA methylation but also chromatin modification, has become an important target for lung cancer research, although previous research has demonstrated that lung cancer develops as a result of both environmental and genetic factors. Here, we demonstrated that an epigenetic regulator/polycomb group protein Bmi1 is more highly expressed in small-cell lung cancer (SCLC) than in non-small-cell lung cancer by immunohistochemical analysis. In vitro experiments indicated that Bmi1 reduction by lentivirus-derived shRNA significantly suppressed proliferation, colony formation and in vivo tumor formation. Importantly, apoptosis was induced by Bmi1 depletion in small-cell lung cancer cells. Furthermore, a tumor suppressor WWOX was identified as a Bmi1 target in the cells by a chromatin immunoprecipitation assay and a quantitative real-time PCR assay; WWOX had a role as a tumor suppressor in SCLC cells; therefore, the Bmi1/WWOX pathway could be a new candidate for a new therapeutic approach for SCLC.

Abstract: Growing evidence has demonstrated that clonogenic cancer stem (initiating) cells are responsible for tumor regrowth and disease relapse. Bmi-1 plays a critical role in the self-renewal of adult stem cells. The Bmi-1 protein is elevated in many types of cancers, and experimental reduction of Bmi-1 protein levels by small interfering RNA (siRNA) causes apoptosis and/or senescence in tumor cells in vitro and increases susceptibility to cytotoxic agents. The Bmi-1 protein has no known enzymatic activity, but serves as the key regulatory component of the PRC1 complex (polycomb repressive complex-1). This complex influences chromatin structure and regulates transcriptional activity of a number of important loci including the Ink4a locus which encodes the tumor suppressor proteins p16(Ink4a) and p14(Arf) . In this prospective study, we will discuss the implication of BMI1 in cancers, the biology of BMI1, and the regulatory control of BMI1 expression. The target validation and the future prospects of targeting BMI1 in cancer therapy are also discussed.

Abstract: The polycomb group (PcG) proteins, particularly Bmi1, have an essential role in maintaining the self-renewing capacity of leukemic stem cells (LSCs). Although one of their major targets in LSCs is known to be the Ink4a/Arf tumor suppressor gene locus, the role of PcG proteins in the leukemic reprogramming of target cells into LSCs is not well characterized. In this study, Bmi1(-/-) granulocyte/macrophage progenitors (GMPs) were transformed with the leukemic fusion gene MLL-AF9. Although Bmi1 was not essential to the immortalization of GMPs in vitro, Bmi1(-/-) cells showed enhanced differentiation and retained less LSCs. A number of genes were derepressed in the absence of Bmi1 including potential tumor suppressor genes. Transplantation assays demonstrated that Bmi1 was indispensable for the development of leukemia in vivo and deletion of both the Ink4a and Arf genes only partially restored the leukemogenic capacity of Bmi1(-/-) LSCs. Of note, the complementation of immortalized Bmi1(-/-)Ink4a-Arf(-/-) GMPs with Bmi1 failed to restore the expression of the majority of deregulated genes and leukemogenic activity in vivo. These findings indicate that Bmi1 is essential for the faithful reprogramming of myeloid progenitors into LSCs and unveil that leukemic fusion genes require PcG proteins exerting an effect in concert to establish LSC-specific transcriptional profiles, which confer full leukemogenic activity on LSCs.