Carboplatin remains a first-line agent in the management of epithelial ovarian cancer (EOC). Unfortunately, platinum-resistant disease ultimately occurs in most patients. Using a novel EOC cell line with acquired resistance to carboplatin: PEO1CarbR, genome-wide micro-array profiling identified the cyclin-dependent kinase inhibitor p57(Kip2) as specifically downregulated in carboplatin resistance. Presently, we describe confirmation of these preliminary data with a variety of approaches.

P27 Kip1 (p27) can prevent cell proliferation by inactivating cyclin-dependent kinases. This function is impaired upon phosphorylation of p27 at tyrosine residue 88. We observed that FLT3 and FLT3-ITD can directly bind and selectively phosphorylate p27 on this residue. Inhibition of FLT3-ITD in cell lines strongly reduced p27 tyrosine 88 phosphorylation and resulted in increased p27 levels and cell cycle arrest. Subsequent analysis revealed the presence of tyrosine 88 phosphorylated p27 in primary patient samples. Inhibition of FLT3 kinase activity with AC220 significantly reduced p27 tyrosine 88 phosphorylation in cells isolated from FLT3 wild type expressing acute myeloid leukemia (AML) patients. In FLT3-ITD positive AML patients, p27 tyrosine 88 phosphorylation was reduced in 5 out of 9 subjects, but, surprisingly, was increased in 4 patients. This indicated that other tyrosine kinases such as Src family kinases might contribute to p27 tyrosine 88 phosphorylation in FLT3-ITD positive AML cells. In fact, incubation with the Src family kinase inhibitor dasatinib could decrease p27 tyrosine 88 phosphorylation in these patient samples, indicating that p27 phosphorylated on tyrosine 88 may be a therapeutic marker for the treatment of AML patients with tyrosine kinase inhibitors.

Sik1 (salt inducible kinase 1) is a serine/threonine kinase that belongs to the stress- and energy-sensing AMP-activated protein kinase family. During murine embryogenesis, sik1 marks the monolayer of future myocardial cells that will populate first the primitive ventricle, and later the primitive atrium suggesting its involvement in cardiac cell differentiation and/or heart development. Despite that observation, the involvement of sik1 in cardiac differentiation is still unknown. We examined the sik1 function during cardiomyocyte differentiation using the ES-derived embryoid bodies. We produced a null embryonic stem cell using a gene-trap cell line carrying an insertion in the sik1 locus. In absence of the sik1 protein, the temporal appearance of cardiomyocytes is delayed. Expression profile analysis revealed sik1 as part of a genetic network that controls the cell cycle, where the cyclin-dependent kinase inhibitor p57(Kip2) is directly involved. Collectively, we provided evidence that sik1-mediated effects are specific for cardiomyogenesis regulating cardiomyoblast cell cycle exit toward terminal differentiation.

The p57(Kip2) cyclin-dependent kinase inhibitor (CDKi) has been implicated in embryogenesis, stem-cell senescence and pathologies, but little is known of its role in cell cycle control. Here, we show that p57(Kip2) is targeted by the p38 stress-activated protein kinase (SAPK). Phosphorylation of p57(Kip2) at T143 by p38 enhances its association with and inhibition of Cdk2, which results in cell-cycle delay upon stress. Genetic inactivation of the SAPK or the CDKi abolishes cell-cycle delay upon osmostress and results in decreased cell viability. Oxidative stress and ionomycin also induce p38-mediated phosphorylation of p57 and cells lacking p38 or p57 display reduced viability to these stresses. Therefore, cell survival to various stresses depends on p57 phosphorylation by p38 that inhibits CDK activity. Together, these findings provide a novel molecular mechanism by which cells can delay cell cycle progression to maximize cell survival upon stress.

p57 (Kip2, cyclin-dependent kinase inhibitor 1C), often found downregulated in cancer, is reported to hold tumor suppressor properties. Originally described as a cyclin-dependent kinase (cdk) inhibitor, p57(KIP2) has since been shown to influence other cellular processes, beyond cell cycle regulation, including cell death and cell migration. Inhibition of cell migration by p57(KIP2) is attributed to the stabilization of the actin cytoskeleton through the activation of LIM domain kinase-1 (LIMK-1). Furthermore, p57(KIP2) is able to enhance mitochondrial-mediated apoptosis. Here, we report that the cell death promoting effect of p57(KIP2) is linked to its effect on the actin cytoskeleton. Indeed, whereas Jasplakinolide, an actin cytoskeleton-stabilizing agent, mimicked p57(KIP2)'s pro-apoptotic effect, destabilizing the actin cytoskeleton with cytochalsin D reversed p57(KIP2)'s pro-apoptotic function. Conversely, LIMK-1, the enzyme mediating p57(KIP2)'s effect on the actin cytoskeleton, was required for p57(KIP2)'s death promoting effect. Finally, p57(KIP2-)mediated stabilization of the actin cytoskeleton was associated with the displacement of hexokinase-1, an inhibitor of the mitochondrial voltage-dependent anion channel, from the mitochondria, providing a possible mechanism for the promotion of the mitochondrial apoptotic cell death pathway. Altogether, our findings link together two tumor suppressor properties of p57(KIP2), by showing that the promotion of cell death by p57(KIP2) requires its actin cytoskeleton stabilization function.

Cell cycle regulators play critical roles in the balance between hematopoietic stem cell (HSC) dormancy and proliferation. In this study, we report that cell cycle entry proceeded normally in HSCs null for cyclin-dependent kinase (CDK) inhibitor p57 due to compensatory upregulation of p27. HSCs null for both p57 and p27, however, were more proliferative and had reduced capacity to engraft in transplantation. We found that heat shock cognate protein 70 (Hsc70) interacts with both p57 and p27 and that the subcellular localization of Hsc70 was critical to maintain HSC cell cycle kinetics. Combined deficiency of p57 and p27 in HSCs resulted in nuclear import of an Hsc70/cyclin D1 complex, concomitant with Rb phosphorylation, and elicited severe defects in maintaining HSC quiescence. Taken together, these data suggest that regulation of cytoplasmic localization of Hsc70/cyclin D1 complex by p57 and p27 is a key intracellular mechanism in controlling HSC dormancy.

CDKN1C (encoding tumor suppressor p57(KIP2)) is a cyclin-dependent kinase (CDK) inhibitor whose family members are often transcriptionally downregulated in human cancer via promoter DNA methylation. In this study, we show that CDKN1C is repressed in breast cancer cells mainly through histone modifications. In particular, we show that CDKN1C is targeted by histone methyltransferase EZH2-mediated histone H3 lysine 27 trimethylation (H3K27me3), and can be strongly activated by inhibition of EZH2 in synergy with histone deacetylase inhibitor. Consistent with the overexpression of EZH2 in a variety of human cancers including breast cancer, CDKN1C in these cancers is downregulated, and breast tumors expressing low levels of CDKN1C are associated with a poor prognosis. We further show that assessing both EZH2 and CDKN1C expression levels as a measurement of EZH2 pathway activity provides a more predictive power of disease outcome than that achieved with EZH2 or CDKN1C alone. Taken together, our study reveals a novel epigenetic mechanism governing CDKN1C repression in breast cancer. Importantly, as a newly identified EZH2 target with prognostic value, it has implications in patient stratification for cancer therapeutic targeting EZH2-mediated gene repression.

Chronic Myeloid Leukemia (CML) is sustained by a small population of cells with stem cell characteristics known as Leukemic Stem Cells that are positive to BCR-ABL fusion protein, involved with several abnormalities in cell proliferation, expansion, apoptosis and cell cycle regulation. Current treatment options for CML involve the use of Tirosine Kinase Inhibitor (Imatinib, Nilotinib and Dasatinib), that efficiently reduce proliferation proliferative cells but do not kill non proliferating CML primitive cells that remain and contributes to the persistence of the disease. In order to understand the role of Cyclin Dependent Kinase Inhibitors in CML LSC permanence after TKI treatment, in this study we analyzed cell cycle status, the levels of several CDKIs and the subcellular localization of such molecules in different CML cell lines, as well as primary CD34(+)CD38(-)lin(-) LSC and HSC. Our results demonstrate that cellular location of p18(INK4c) and p57(Kip2) seems to be implicated in the antiproliferative activity of Imatinib and Dasatinib in CML cells and also suggest that the permanence of quiescent stem cells after TKI treatment could be associated with a decrease in p18(INK4c) and p57(Kip2) nuclear location. The differences in p18(INK4c)and p57(Kip2)activities in CML and normal stem cells suggest a different cell cycle regulation and provide a platform that could be considered in the development of new therapeutic options to eliminate LSC.

The eukaryotic cell cycle is negatively regulated by cyclin-dependent kinase inhibitors (CKIs). p57Kip2 is a member of the Cip/Kip family of CKIs and frequently inactivated by genomic mutations associated with human overgrowth disorders. There is increasing evidence for p57 to control cellular processes in addition to cell cycle and CDK regulation including transcription, apoptosis, migration or development. In order to obtain molecular insights to unknown functions of p57, we performed a protein interaction screen. We identified the transcription regulator four-and-a-half LIM-only protein 2 (FHL2) as a novel p57-binding protein. Co-immunoprecipitation and reporter gene assays were used to elucidate the physiological and functional relevance of p57/FHL2 interaction. We found in cancer cells that endogenous p57 and FHL2 are in a complex. We observed a substantial induction of established FHL2-regulated gene promoters by p57 in reporter gene experiments and detected strong induction of the intrinsic transactivation activity of FHL2. Treatment of cells with histone deacetylase (HDAC) inhibitors and binding of exogenous FHL2 to HDACs indicated repression of FHL2 transcription activity by HDACs. In the presence of the HDAC inhibitor sodium butyrate activation of FHL2 by p57 is abrogated suggesting that p57 shares a common pathway with HDAC inhibitors. p57 competes with HDACs for FHL2 binding which might partly explain the mechanism of FHL2 activation by p57. These results suggest a novel function of p57 in transcription regulation.

Adult skeletal muscle maintenance and regeneration depend on efficient muscle stem cell (MuSC) functions. The mechanisms coordinating cell cycle with activation, renewal, and differentiation of MuSCs remain poorly understood. Here, we investigated how adult MuSCs are regulated by CDKN1c (p57kip2), a cyclin-dependent kinase inhibitor, using mouse molecular genetics. In the absence of CDKN1c, skeletal muscle repair is severely impaired after injury. We show that CDKN1c is not expressed in quiescent MuSCs, while being induced in activated and proliferating myoblasts and maintained in differentiating myogenic cells. In agreement, isolated Cdkn1c-deficient primary myoblasts display differentiation defects and increased proliferation. We further show that the subcellular localization of CDKN1c is dynamic; while CDKN1c is initially localized to the cytoplasm of activated/proliferating myoblasts, progressive nuclear translocation leads to growth arrest during differentiation. We propose that CDKN1c activity is restricted to differentiating myoblasts by regulated cyto-nuclear relocalization, coordinating the balance between proliferation and growth arrest.

Although the mammalian intestinal epithelium manifests robust regenerative capacity after various cytotoxic injuries, the underlying mechanism has remained unclear. Here we identify the cyclin-dependent kinase inhibitor p57 as a specific marker for a quiescent cell population located around the +4 position of intestinal crypts. Lineage tracing reveals that the p57+ cells serve as enteroendocrine/tuft cell precursors under normal conditions but dedifferentiate and act as facultative stem cells to support regeneration after injury. Single-cell transcriptomics analysis shows that the p57+ cells undergo a dynamic reprogramming process after injury that is characterized by fetal-like conversion and metaplasia-like transformation. Population-level analysis also detects such spatiotemporal reprogramming widely in other differentiated cell types. In intestinal adenoma, p57+ cells manifest homeostatic stem cell activity, in the context of constitutively activated spatiotemporal reprogramming. Our results highlight a pronounced plasticity of the intestinal epithelium that supports maintenance of tissue integrity in normal and neoplastic contexts.

Adult neurogenesis is supported by multipotent neural stem cells (NSCs) with unique properties and growth requirements. Adult NSCs constitute a reversibly quiescent cell population that can be activated by extracellular signals from the microenvironment in which they reside in vivo. Although genomic imprinting plays a role in adult neurogenesis through dose regulation of some relevant signals, the roles of many imprinted genes in the process remain elusive. Insulin-like growth factor 2 (IGF2) is encoded by an imprinted gene that contributes to NSC maintenance in the adult subventricular zone through a biallelic expression in only the vascular compartment. We show here that IGF2 additionally promotes terminal differentiation of NSCs into astrocytes, neurons and oligodendrocytes by inducing the expression of the maternally expressed gene cyclin-dependent kinase inhibitor 1c (Cdkn1c), encoding the cell cycle inhibitor p57. Using intraventricular infusion of recombinant IGF2 in a conditional mutant strain with Cdkn1c-deficient NSCs, we confirm that p57 partially mediates the differentiation effects of IGF2 in NSCs and that this occurs independently of its role in cell-cycle progression, balancing the relationship between astrogliogenesis, neurogenesis and oligodendrogenesis.

Germline inactivation of the von Hippel-Lindau (VHL) tumor suppressor predisposes patients to develop different highly vascularized cancers. pVHL targets the hypoxia-inducible transcription factor (HIF-1α) for degradation, modulating the activation of various genes involved in hypoxia response. Hypoxia plays a relevant role in regulating cell cycle progression, inducing growth arrest in cells exposed to prolonged oxygen deprivation. However, the exact molecular details driving this transition are far from understood. Here, we present novel interactions between pVHL and the cyclin-dependent kinase inhibitor family CDKN1 (p21, p27 and p57). Bioinformatics analysis, yeast two-hybrid screening and co-immunoprecipitation assays were used to predict, dissect and validate the interactions. We found that the CDKN1 proteins share a conserved region mimicking the HIF-1α motif responsible for pVHL binding. Intriguingly, a p27 site-specific mutation associated to cancer is shown to modulate this novel interaction. Our findings suggest a new connection between the pathways regulating hypoxia and cell cycle progression.

Loss of genomic imprinting is involved in a number of developmental abnormalities and cancers. ZAC is an imprinted gene expressed from the paternal allele of chromosome 6q24 within a region known to harbor a tumor suppressor gene for several types of neoplasia. p57(KIP2) (CDKN1C) is a maternally expressed gene located on chromosome 11p15.5 which encodes a cyclin-dependent kinase inhibitor that may also act as a tumor suppressor gene. Mutations in ZAC and p57KIP2 have been implicated in transient neonatal diabetes mellitus (TNDB) and Beckwith-Wiedemann syndrome, respectively. Patients with these diseases share many characteristics. Here we show that mouse Zac1 and p57Kip2 have a strikingly similar expression pattern. ZAC, a sequence-specific DNA-binding protein, binds within the CpG island of LIT1 (KCNQ1OT1), a paternally expressed, anti-sense RNA thought to negatively regulate p57(KIP2) in cis. ZAC induces LIT1 transcription in a methylation-dependent manner. Our data suggest that ZAC may regulate p57(KIP2) through LIT1, forming part of a novel signaling pathway regulating cell growth. Mutations in ZAC may, therefore, contribute to Beckwith-Wiedemann syndrome. Furthermore, we find changes in DNA methylation at the LIT1 putative imprinting control region in two patients with TNDB.

The p53 family comprises the tumor suppressor p53 and the structural homologs p63 and p73. How the three family members cooperate in tumor suppression remains unclear. Here, we report different but complementary functions of the individual members for regulating retinoblastoma protein (RB) function during myogenic differentiation. Whereas p53 transactivates the retinoblastoma gene, p63 and p73 induce the cyclin-dependent kinase inhibitor p57 to maintain RB in an active, hypophosphorylated state. DeltaNp73 inhibits these functions of the p53 family in differentiation control, prevents myogenic differentiation, and enables cooperating oncogenes to transform myoblasts to tumorigenicity. DeltaNp73 is frequently overexpressed in rhabdomyosarcoma and essential for tumor progression in vivo. These findings establish differentiation control as a key tumor suppressor activity of the p53 family.

Myelosuppression is a life-threatening complication of antineoplastic therapy, but treatment is restricted to a few cytokines with unilineage hematopoietic activity. Although hematopoietic stem cells (HSCs) are predominantly quiescent during homeostasis, they are rapidly recruited into cell cycle by stresses, including myelosuppressive chemotherapy. Factors that induce HSCs to proliferate during stress have been characterized, but it is not known how HSC quiescence is then reestablished. In this study, we show that TGFβ signaling is transiently activated in hematopoietic stem and progenitor cells (HSPCs) during hematopoietic regeneration. Blockade of TGFβ signaling after chemotherapy accelerates hematopoietic reconstitution and delays the return of cycling HSCs to quiescence. In contrast, TGFβ blockade during homeostasis fails to induce cycling of HSPCs. We identified the cyclin-dependent kinase inhibitor Cdkn1c (p57) as a key downstream mediator of TGFβ during regeneration because the recovery of chimeric mice, incapable of expressing p57 in HSPCs, phenocopies blockade of TGFβ signaling after chemotherapy. This study demonstrates that context-dependent activation of TGFβ signaling is central to an unrecognized counterregulatory mechanism that promotes homeostasis once hematopoiesis has sufficiently recovered from myelosuppressive chemotherapy. These results open the door to new, potentially superior, approaches to promote multilineage hematopoietic recovery by blocking the TGFβ signaling that dampens regeneration.

Although 19p13.13 microdeletion syndrome has been consistently associated with intellectual disability, overgrowth, and macrocephaly, the underlying mechanisms remain unclear. MAST1, a member of the microtubule-associated serine/threonine kinase family, has been suggested as a potential candidate gene responsible for neurologic abnormalities in 19p13.13 microdeletion syndrome, but its role in nervous system development remains to be elucidated. Here, we investigated how MAST1 contributes to neuronal development. We report that MAST1 is upregulated during neuronal differentiation of the human neuroblastoma cell line, SH-SY5Y. Inhibition of MAST1 expression by RNA interference attenuated neuronal differentiation of SH-SY5Y cells. Cell cycle analyses revealed that MAST1-depleted cells did not undergo cell cycle arrest after RA treatment. Consistent with this observation, the number of EdU-positive cells significantly increased in MAST1 knockdown cells. Intriguingly, levels of P27, a cyclin-dependent kinase inhibitor, were also increased during neuronal differentiation, and MAST1 knockdown reduced the expression of P27. Moreover, reduced neuronal differentiation caused by MAST1 depletion was rescued partially by P27 overexpression in SH-SY5Y cells. Collectively, these results suggest that MAST1 influences nervous system development by affecting neuronal differentiation through P27.

Glucocorticoids (GCs) affect the proliferation of growth plate chondrocytes. In this study, we investigated the role of the GC-inducible promyelocytic leukemia zinc finger (PLZF) gene in chondrocyte differentiation by using the chondrogenic cell line ATDC5. PLZF overexpression suppressed cell cycle progression (p < 0.01) and promoted differentiation into hypertrophic chondrocytes by inducing mRNA expression of alkaline phosphatase (p < 0.01), and the cyclin-dependent kinase (CDK) inhibitor p21 (p < 0.01). In contrast, PLZF knockdown impaired differentiation into hypertrophic chondrocytes and promoted cell cycle progression (p < 0.01). Treatment with the GC analogue dexamethasone (10(-6) M) suppressed cell cycle progression in ATDC5 cells. PLZF shRNA attenuated dexamethasone-induced cell cycle arrest (p < 0.01) by downregulating the mRNA expression of the CDK inhibitors p21 and p57 (p < 0.01). These results clearly indicated that PLZF promoted differentiation into hypertrophic chondrocytes and mediated dexamethasone-induced cell cycle arrest by regulating CDK inhibitors.

Glomerular hypertrophy is a crucial factor of severe podocyte damage and proteinuria. Our previous study showed that high fructose induced podocyte injury. The current study aimed to explore a novel molecular mechanism underlying podocyte hypertrophy induced by high fructose. Here we demonstrated for the first time that high fructose significantly initiated the hypertrophy in rat glomeruli and differentiated human podocytes (HPCs). Consistently, it induced inflammatory response with the down-regulation of anti-inflammatory factor zinc-finger protein tristetraprolin (TTP) and the activation of interleukin-6 (IL-6)/signal transducer and activator of transcription 3 (STAT3) signaling in these animal and cell models. Subsequently, high-expression of microRNA-92a-3p (miR-92a-3p) and its target protein cyclin-dependent kinase inhibitor p57 (P57) down-regulation, representing abnormal proliferation and apoptosis, were observed in vivo and in vitro. Moreover, high fructose increased ketohexokinase-A (KHK-A) expression in rat glomeruli and differentiated HPCs. Exogenous IL-6 stimulation up-regulated IL-6/STAT3 signaling and miR-92a-3p, reduced P57 expression and promoted podocyte proliferation, apoptosis and hypertrophy in vitro. The data from anti-inflammatory agent maslinic acid treatment or TTP siRNA transfection showed that high fructose may decrease TTP to activate IL-6/STAT3 signaling in podocyte overproliferation and apoptosis, causing podocyte hypertrophy. Whereas, KHK-A siRNA transfection remarkably restored high fructose-induced TTP down-regulation, IL-6/STAT3 signaling activation, podocyte overproliferation, apoptosis and hypertrophy in differentiated HPCs. Taken together, these results suggested that high fructose possibly increased KHK-A expression to down-regulate TTP, subsequently activated IL-6/STAT3 signaling to interfere with podocyte proliferation and apoptosis by up-regulating miR-92a-3p to suppress P57 expression, causing podocyte hypertrophy. Therefore, the inactivation of IL-6/STAT3 to relieve podocyte hypertrophy mediated by inhibiting KHK-A to increase TTP may be a novel strategy for high fructose diet-associated podocyte injury and proteinuria.

Phenethyl isothiocyanate (PEITC), a component in cruciferous vegetables, can block chemical carcinogenesis in animal models. Our objective was to determine the effect of treatment with PEITC on gene expression changes in MCF-7 human breast cancer cells in order to evaluate potential mechanisms involved in its chemopreventive effects. MCF-7 cells were treated for 48 hours with either PEITC (3 μM) or the vehicle. Total RNA was extracted from cell membrane preparations, and labeled cDNA's representing the mRNA pool were reverse-transcribed directly from total RNA isolated for use in the microarray hybridizations. Two specific human GE Array Kits (Superarray Inc.) that both contain 23 marker genes, related to signal transduction pathways or cancer/tumor suppression, plus 2 housekeeping genes (β-actin and GAPDH), were utilized. Arrays from treated and control cells (n = 4 per group) were evaluated using a Student's t-test. Gene expression was significantly induced for tumor protein p53 (p53), cyclin-dependent kinase inhibitor 1C (p57 Kip2), breast cancer Type 2 early onset (BRCA2), cAMP responsive element binding protein 2 (ATF-2), interleukin 2 (IL-2), heat shock 27 KD protein (hsp27), and CYP19 (aromatase). Induction of p57 Kip2, p53, BRCA2, IL-2, and ATF-2 would be expected to decrease cellular proliferation and increase tumor suppression and/or apoptosis. PEITC treatment produced significant alterations in some genes involved in tumor suppression and cellular proliferation/apoptosis that may be important in explaining the chemopreventive effects of PEITC.