The Sin3-histone deacetylase (HDAC) corepressor complex is conserved from yeast to humans. Mammals possess two highly related Sin3 proteins, mSin3A and mSin3B, which serve as scaffolds tethering HDAC enzymatic activity, and numerous sequence-specific transcription factors to enable local chromatin regulation at specific gene targets. Despite broad overlapping expression of mSin3A and mSin3B, mSin3A is cell-essential and vital for early embryonic development. Here, genetic disruption of mSin3B reveals a very different phenotype characterized by the survival of cultured cells and lethality at late stages of embryonic development with defective differentiation of multiple lineages-phenotypes that are strikingly reminiscent of those associated with loss of retinoblastoma family members or E2F transcriptional repressors. Additionally, we observe that, whereas mSin3B ؊/؊ cells cycle normally under standard growth conditions, they show an impaired ability to exit the cell cycle with limiting growth factors. Correspondingly, mSin3B interacts physically with the promoters of known E2F target genes, and its deficiency is associated with derepression of these gene targets in vivo. Together, these results reveal a critical role for mSin3B in the control of cell cycle exit and terminal differentiation in mammals and establish contrasting roles for the mSin3 proteins in the growth and development of specific lineages.E2F ͉ histone deacetylase ͉ knockout ͉ quiescence T he Sin3-histone deacetylase (HDAC) corepressor has been physically and functionally linked to diverse transcriptional complexes governing many physiological processes (1, 2). The two highly related mammalian Sin3 proteins, mSin3A and mSin3B, use their multiple interaction domains to direct chromatin-modifying activities to specific sites in the genome, most typically via sequence-specific transcription factors and their cognate binding elements. Class I HDACs, HDAC1 and HDAC2, are the principal enzymatic activities of the mSin3 complex. In addition, there are several other mSin3-associated proteins, including mSds3, p33 ING1 ,.mSin3A has been shown to be essential for early embryonic development and for the growth and survival of cultured cells that may relate to its requirement for the regulation of multiple transcriptional programs (7). Of relevance to the current study, mSin3B is expressed in cells deleted for mSin3A, suggesting that, despite their structural relatedness, mSin3B is not functionally equivalent to mSin3A. Both yeast and mammalian Sds3 are required for the maintenance of Sin3-associated HDAC enzymatic activity (3,8). Nullizygosity for mammalian Sds3 results in early embryonic lethality and engenders marked chromosome segregation defects due to a failure in pericentric heterochromatin formation (9). mSin3A-null fibroblasts exhibited normal karyotypes (7).In mammalian cell cycle, the G 0 /G 1 -to-S transition is a highly regulated event whose disruption represents a prerequisite for essentially all human cancers. This critical phase of the cell cy...
Embryonal rhabdomyosarcoma (ERMS) is a common pediatric malignancy of muscle, with relapse being the major clinical challenge. Selfrenewing tumor-propagating cells (TPCs) drive cancer relapse and are confined to a molecularly definable subset of ERMS cells. To identify drugs that suppress ERMS self-renewal and induce differentiation of TPCs, a large-scale chemical screen was completed. Glycogen synthase kinase 3 (GSK3) inhibitors were identified as potent suppressors of ERMS growth through inhibiting proliferation and inducing terminal differentiation of TPCs into myosin-expressing cells. In support of GSK3 inhibitors functioning through activation of the canonical WNT/ β-catenin pathway, recombinant WNT3A and stabilized β-catenin also enhanced terminal differentiation of human ERMS cells. Treatment of ERMS-bearing zebrafish with GSK3 inhibitors activated the WNT/ β-catenin pathway, resulting in suppressed ERMS growth, depleted TPCs, and diminished self-renewal capacity in vivo. Activation of the canonical WNT/β-catenin pathway also significantly reduced selfrenewal of human ERMS, indicating a conserved function for this pathway in modulating ERMS self-renewal. In total, we have identified an unconventional tumor suppressive role for the canonical WNT/ β-catenin pathway in regulating self-renewal of ERMS and revealed therapeutic strategies to target differentiation of TPCs in ERMS.
Lung cancer is the most common cause of cancer-related mortality worldwide. Here, we report elevated expression of tribbles homolog 2 (TRIB2) in primary human lung tumors and in non-small cell lung cancer cells that express low levels of differentiation-inducing transcription factor CCAAT/enhancer-binding protein alpha (C/EBPα). In approximately 10–20% of cases, elevated TRIB2 expression resulted from gene amplification. TRIB2 knockdown was found to inhibit cell proliferation and in vivo tumor growth. In addition, TRIB2 knockdown led to morphological changes similar to C/EBPα overexpression and correlated with increased expression and activity of C/EBPα. TRIB2-mediated regulation of C/EBPα was found to occur through the association of TRIB2 with the E3 ligase TRIM21. Together, these data identify TRIB2 as a potential driver of lung tumorigenesis through a mechanism that involves downregulation of C/EBPα.
Serial passage of primary mammalian cells or strong mitogenic signals induce a permanent exit from the cell cycle called senescence. A characteristic of senescent cells is the heterochromatinization of loci encoding pro-proliferative genes, leading to their transcriptional silencing. Senescence is thought to represent a defense mechanism against uncontrolled proliferation and cancer. Consequently, genetic alterations that allow senescence bypass are associated with susceptibility to oncogenic transformation. We show that fibroblasts genetically inactivated for the chromatin-associated Sin3B protein are refractory to replicative and oncogene-induced senescence. Conversely, overexpression of Sin3B triggers senescence and the formation of senescence-associated heterochromatic foci. Although Sin3B is strongly up-regulated upon oncogenic stress, decrease in expression of Sin3B is associated with tumor progression in vivo, suggesting that expression of Sin3B may represent a barrier against transformation. Together, these results underscore the contribution of senescence in tumor suppression and suggest that expression of chromatin modifiers is modulated at specific stages of cellular transformation. Consequently, these findings suggest that modulation of Sin3B-associated activities may represent new therapeutic opportunities for treatment of cancers. [Cancer Res 2009;69(16):6430-7]
Purpose Anaplastic Lymphoma Kinase (ALK) is the most frequently mutated oncogene in the pediatric cancer neuroblastoma. We performed an in vitro screen for synergistic drug combinations that target neuroblastomas with mutations in ALK to determine if drug combinations could enhance anti-tumor efficacy. Experimental Design We screened combinations of eight molecularly targeted agents against seventeen comprehensively characterized human neuroblastoma-derived cell lines. We investigated the combination of Ceritinib and Ribociclib on in vitro proliferation, cell cycle, viability, caspase activation, and the Cyclin D/CDK4/CDK6/RB and pALK signaling networks in cell lines with representative ALK status. We performed in vivo trials in CB17 SCID mice bearing conventional and patient-derived xenograft models comparing Ceritinib alone, Ribociclib alone, and the combination, with plasma pharmacokinetics to evaluate for drug-drug interactions. Results The combination of Ribociclib, a dual inhibitor of cyclin-dependent kinase (CDK) 4 and 6, and the ALK inhibitor Ceritinib demonstrated higher cytotoxicity (p=0.008) and synergy scores (p=0.006) in cell lines with ALK mutations as compared to cell lines lacking mutations or alterations in ALK. Compared to either drug alone, combination therapy enhanced growth inhibition, cell cycle arrest, and caspase-independent cell death. Combination therapy achieved complete regressions in neuroblastoma xenografts with ALK-F1174L and F1245C de novo resistance mutations, and prevented the emergence of resistance. Murine Ribociclib and Ceritinib plasma concentrations were unaltered by combination therapy. Conclusions This preclinical combination drug screen with in vivo validation has provided the rationale for a first in children trial of combination Ceritinib and Ribociclib in a molecularly selected pediatric population.
Emerging evidence suggests that some cancers contain a population of stem-like TICs (tumor-initiating cells) and eliminating TICs may offer a new strategy to develop successful anti-cancer therapies. As molecular mechanisms underlying the maintenance of the TIC pool are poorly understood, the development of TIC-specific therapeutics remains a major challenge. We first identified and characterized TICs and non-TICs isolated from a mouse breast cancer model. TICs displayed increased tumorigenic potential, self-renewal, heterogeneous differentiation, and bipotency. Gene expression analysis and immunostaining of TICs and non-TICs revealed that FGFR2 was preferentially expressed in TICs. Loss of FGFR2 impaired self-renewal of TICs, thus resulting in marked decreases in the TIC population and tumorigenic potential. Restoration of FGFR2 rescued the defects in TIC pool maintenance, bipotency, and breast tumor growth driven by FGFR2 knockdown. In addition, pharmacological inhibition of FGFR2 kinase activity led to a decrease in the TIC population which resulted in suppression of breast tumor growth. Moreover, human breast TICs isolated from patient tumor samples were found enriched in a FGFR2+ population that was sufficient to initiate tumor growth. Our data suggest that FGFR2 is essential in sustaining the breast TIC pool through promotion of self-renewal and maintenance of bipotent TICs, and raise the possibility of FGFR2 inhibition as a strategy for anti-cancer therapy by eradicating breast TICs.
Efficient and accurate cell cycle exit is intimately linked to cellular differentiation, and by inference, to the prevention of tumorigenesis. Perhaps the most important axis of control for this process involves the interactions of the E2F family of DNA binding proteins with the retinoblastoma (Rb) and Rb-related "pocket protein" (p107 and p130) family of tumor suppressors. Not surprisingly, alterations in this pathway are present in a large number of human malignancies. The molecular basis for the controls exercised by the Rb family of proteins has been widely investigated, but is still not completely understood. Elegant in vitro studies had previously suggested the participation of histone deacetylase (HDAC)-associated Sin3B in E2F-mediated repression. Using genetically modified mice, we have recently uncovered a role for the Sin3B protein as a specific and essential actor in promoting cell cycle exit via the E2F-Rb pathway. We demonstrated its absolute requirement not only for cell cycle exit in vitro and in vivo, but also for biological processes linked to cellular differentiation. These observations strongly suggest that Sin3B plays an essential role in coordinating the chromatin modifying activities required for the transient repression of pro-proliferation genes in quiescence, as well as stable silencing of these genes upon terminal differentiation.
BackgroundNeuroblastoma (NB) is the most common extracranial solid tumor in children. NB tumors and derived cell lines are phenotypically heterogeneous. Cell lines are classified by phenotype, each having distinct differentiation and tumorigenic properties. The neuroblastic phenotype is tumorigenic, has neuronal features and includes stem cells (I-cells) and neuronal cells (N-cells). The non-neuronal phenotype (S-cell) comprises cells that are non-tumorigenic with features of glial/smooth muscle precursor cells. This study identified miRNAs associated with each distinct cell phenotypes and investigated their role in regulating associated differentiation and tumorigenic properties.MethodsA miRNA microarray was performed on the three cell phenotypes and expression verified by qRT-PCR. miRNAs specific for certain cell phenotypes were modulated using miRNA inhibitors or stable transfection. Neuronal differentiation was induced by RA; non-neuronal differentiation by BrdU. Changes in tumorigenicity were assayed by soft agar colony forming ability. N-myc binding to miR-375 promoter was assayed by chromatin-immunoprecipitation.ResultsUnsupervised hierarchical clustering of miRNA microarray data segregated neuroblastic and non-neuronal cell lines and showed that specific miRNAs define each phenotype. qRT-PCR validation confirmed that increased levels of miR-21, miR-221 and miR-335 are associated with the non-neuronal phenotype, whereas increased levels of miR-124 and miR-375 are exclusive to neuroblastic cells. Downregulation of miR-335 in non-neuronal cells modulates expression levels of HAND1 and JAG1, known modulators of neuronal differentiation. Overexpression of miR-124 in stem cells induces terminal neuronal differentiation with reduced malignancy. Expression of miR-375 is exclusive for N-myc-expressing neuroblastic cells and is regulated by N-myc. Moreover, miR-375 downregulates expression of the neuronal-specific RNA binding protein HuD.ConclusionsThus, miRNAs define distinct NB cell phenotypes. Increased levels of miR-21, miR-221 and miR-335 characterize the non-neuronal, non-malignant phenotype and miR-335 maintains the non-neuronal features possibly by blocking neuronal differentiation. miR-124 induces terminal neuronal differentiation with reduction in malignancy. Data suggest N-myc inhibits neuronal differentiation of neuroblastic cells possibly by upregulating miR-375 which, in turn, suppresses HuD. As tumor differentiation state is highly predictive of patient survival, the involvement of these miRNAs with NB differentiation and tumorigenic state could be exploited in the development of novel therapeutic strategies for this enigmatic childhood cancer.
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