Miki Obata

p62/Sqstm1 promotes malignancy of HCV-positive hepatocellular carcinoma through Nrf2-dependent metabolic reprogramming

p62/Sqstm1 is a multifunctional protein involved in cell survival, growth and death, that is degraded by autophagy. Amplification of the p62/Sqstm1 gene, and aberrant accumulation and phosphorylation of p62/Sqstm1, have been implicated in tumour development. Herein, we reveal the molecular mechanism of p62/Sqstm1-dependent malignant progression, and suggest that molecular targeting of p62/Sqstm1 represents a potential chemotherapeutic approach against hepatocellular carcinoma (HCC). Phosphorylation of p62/Sqstm1 at Ser349 directs glucose to the glucuronate pathway, and glutamine towards glutathione synthesis through activation of the transcription factor Nrf2. These changes provide HCC cells with tolerance to anti-cancer drugs and proliferation potency. Phosphorylated p62/Sqstm1 accumulates in tumour regions positive for hepatitis C virus (HCV). An inhibitor of phosphorylated p62-dependent Nrf2 activation suppresses the proliferation and anticancer agent tolerance of HCC. Our data indicate that this Nrf2 inhibitor could be used to make cancer cells less resistant to anticancer drugs, especially in HCV-positive HCC patients.

Lack of Bcl11b tumor suppressor results in vulnerability to DNA replication stress and damages

Bcl11b/Rit1 is involved in T-cell development and undergoes chromosomal rearrangements in human T-cell leukemias. Thymocytes of Bcl11b−/− newborn mice exhibit apoptosis at a certain developmental stage when thymocytes re-enter into the cell-cycle. Here, we show that Bcl11b-knockdown T-cell lines, when exposed to growth stimuli, exhibited apoptosis at the S phase with concomitant decreases in a cell-cycle inhibitor, p27 and an antiapoptotic protein, Bcl-xL, owing to transcriptional repression. This repression was a likely consequence of the impairment of Sirt1, a nicotinamide adenine dinucleotide-dependent deacetylase associating with Bcl11b. Activation of the apoptotic process cleaved the mediator protein, Claspin, and inhibited phosphorylation of cell-cycle checkpoint kinase 1 (Chk1) that plays a central role in sensing and responding to incomplete replication. Bcl11b−/− thymocytes also failed to phosphorylate Chk1 when UV irradiated. These results implicate Bcl11b in the remedy for DNA replication stress and maintenance of genomic integrity.

Predisposition to mouse thymic lymphomas in response to ionizing radiation depends on variant alleles encoding metal-responsive transcription factor-1 (Mtf-1 )

Genetic predisposition to cancers is significant to public health because a high proportion of cancers probably arise in a susceptible human subpopulation. Using a mouse model of γ-ray-induced thymic lymphomas, we performed linkage analysis and haplotype mapping that suggested Mtf-1, metal-responsive transcription factor-1 (Mtf-1), as a candidate lymphoma susceptibility gene. Sequence analysis revealed a polymorphism of Mtf-1 that alters the corresponding amino acid at position 424 in the proline-rich domain from a serine in susceptibility strains to proline in resistant strains. The transcriptional activity of Mtf-1 encoding serine and proline was compared by transfecting the DNA to Mtf-1-null cells, and the change to proline conferred a higher metal responsiveness in transfections. Furthermore, the resistant congenic strains possessing the Mtf-1 allele of proline type exhibited higher radiation inducibility of target genes than susceptible background strains having the Mtf-1 allele of serine type. Since products of the targets such as metallothionein are able to suppress cellular stresses generated by irradiation, these results suggest that highly inducible strains having Mtf-1 of proline type are refractory to radiation effects and hence are resistant to lymphoma development.

Bcl11b transcription factor plays a role in the maintenance of the ameloblast-progenitors in mouse adult maxillary incisors

Rodent incisors maintain the ability to grow continuously and their labial dentin is covered with enamel. Bcl11b zinc-finger transcription factor is expressed in ameloblast progenitors in mouse incisors and its absence in Bcl11b(KO/KO) mice results in a defect in embryonic tooth development. However, the role of Bcl11b in incisor maintenance in adult tissue was not studied because of death at birth in Bcl11b(KO/KO) mice. Here, we examined compound heterozygous Bcl11b(S826G/KO) mice, one allele of which has an amino acid substitution of serine at position 826 for glycine, that exhibited hypoplastic maxillary incisors with lower concentrations of minerals at the enamel and the dentin, accompanying the maxillary bone hypoplasia. Histological examinations revealed hypoplasia of the labial cervical loop in incisors, shortening of the ameloblast progenitor region, and impairment in differentiation and proliferation of ameloblast-lineage cells. Interestingly, however, juvenile mice at 5days after birth did not show marked change in these phenotypes. These results suggest that attenuated Bcl11b activity impairs ameloblast progenitors and incisor maintenance. The number of BrdU label-retaining cells, putative stem cells, was lower in Bcl11b(S826G/KO) incisors, which suggests the incisor hypoplasia may be in part a result of the decreased number of stem cells. Interestingly, the level of Shh and FGF3 expressions, which are assumed to play key roles in the development and maintenance of ameloblasts and odontoblasts, was not decreased, though the expressed areas were more restricted in ameloblast progenitor and mesenchyme regions of Bcl11b(S826G/KO) incisors, respectively. Those data suggest that the incisor maintenance by Bcl11b is not directly related to the FGF epithelial-mesenchymal signaling loop including Shh but is intrinsic to ameloblast progenitors and possibly stem cells.

Bcl11b SWI/SNF-complex subunit modulates intestinal adenoma and regeneration after γ-irradiation through Wnt/β-catenin pathway.

SWI/SNF chromatin remodeling complexes constitute a highly related family of multi-subunit complexes to modulate transcription, and SWI/SNF subunit genes are collectively mutated in 20% of all human cancers. Bcl11b is a SWI/SNF subunit and acts as a haploinsufficient tumor suppressor in leukemia/lymphomas. Here, we show expression of Bcl11b in intestinal crypt cells and promotion of intestinal tumorigenesis by Bcl11b attenuation in Apc (min/+) mice. Of importance, mutations or allelic loss of BCL11B was detected in one-third of human colon cancers. We also show that attenuated Bcl11b activity in the crypt base columnar (CBC) cells expressing the Lgr5 stem cell marker enhanced regeneration of intestinal epithelial cells after the radiation-induced injury. Interestingly, BCL11B introduction in human cell lines downregulated transcription of β-catenin target genes, whereas Bcl11b attenuation in Lgr5(+) CBCs increased expression of β-catenin targets including c-Myc and cyclin D1. Together, our results argue that Bcl11b impairment promotes tumor development in mouse and human intestine at least in part through deregulation of β-catenin pathway.

BCL11B tumor suppressor inhibits HDM2 expression in a p53-dependent manner.

BCL11B is a C2H2 zinc finger transcription factor that acts as a haploinsufficient tumor suppressor. Mutations and deletion in the human orthologue BCL11B have been identified in human T-cell acute lymphoblastic leukemia (T-ALL) and a mouse model of thymic lymphomas. Bcl11b(KO/+)p53(KO/+) doubly heterozygous mice, but not Bcl11b(KO/+) heterozygous mice, spontaneously develop thymic lymphomas at a high frequency, suggesting cooperativity of BCL11B and p53 in cancer development. In this study, we have examined whether or not BCL11B directly affects the p53 signaling pathway including HDM2, a ubiquitin ligase for p53 degradation. The p53 pathway regulates cell proliferation and the response to DNA damages to maintain genome integrity. Here we show that BCL11B binds to human HDM2-P2 promoter by ChIP (chromatin immuno-precipitation) assay and inhibits HDM2 expression in a p53-dependent manner. Deletion of the distal p53 responsive element in HDM2 promoter region or the lack of p53 in HCT116 cells greatly reduced the repressive effect of BCL11B on HDM2-P2 promoter activity. The repressive activity was alleviated in γ-ray induced DNA damage conditions that activate p53, suggesting interaction between BCL11B and p53 for HDM2 expression. These date suggest that BCL11B affects the activity of the p53-HDM2 feedback loop in basal and irradiated conditions. This may be a mechanism underlying the leukemic transformation in T-ALL and in Bcl11b(KO/+)p53(KO/+) mouse thymocytes.

Cell of origin in radiation-induced premalignant thymocytes with differentiation capability in mice conditionally losing one Bcl11b allele

Bcl11b is a haploinsufficient tumor suppressor, mutations or deletion of which has been found in 10–16% of T‐cell acute lymphoblastic leukemias. Bcl11bKO/+ heterozygous mice are susceptible to thymic lymphomas, a model of T‐cell acute lymphoblastic leukemia, when γ‐irradiated, and irradiated Bcl11bKO/+ mice generate clonally expanding or premalignant thymocytes before thymic lymphoma development. Cells with radiation‐induced DNA damages are assumed to be the cells of origin in tumors; however, which thymocyte is the tumor cell origin remains obscure. In this study we generated Bcl11bflox/+;Lck‐Cre and Bcl11bflox/+;CD4‐Cre mice; in the former, loss of one Bcl11b allele occurs in thymocytes at the immature CD4−CD8− stage, whereas in the latter the loss occurs in the more differentiated CD4+CD8+ double‐positive stage. We examined clonal expansion and differentiation of thymocytes in mice 60 days after 3 Gy γ‐irradiation. Half (9/18) of the thymuses in the Bcl11bflox/+;Lck‐Cre group showed limited rearrangement sites at the T‐cell receptor‐β (TCRβ) locus, indicating clonal cell expansion, but none in the Bcl11bflox/+;CD4‐Cre group did. This indicates that the origin of the premalignant thymocytes is not in double‐positive cells but immature thymocytes. Interestingly, those premalignant thymocytes underwent rearrangement at various different sites of the TCRα locus and the majority showed a higher expression of TCRβ and CD8, and more differentiated phenotypes. This suggests the existence of a subpopulation of immature cells within the premalignant cells that is capable of proliferating and continuously producing differentiated thymocytes.

Clonally Expanding Thymocytes Having Lineage Capability in Gamma-Ray–Induced Mouse Atrophic Thymus

PURPOSE To characterize, in the setting of gamma-ray-induced atrophic thymus, probable prelymphoma cells showing clonal growth and changes in signaling, including DNA damage checkpoint. METHODS AND MATERIALS A total of 111 and 45 mouse atrophic thymuses at 40 and 80 days, respectively, after gamma-irradiation were analyzed with polymerase chain reaction for D-J rearrangements at the TCRbeta locus, flow cytometry for cell cycle, and Western blotting for the activation of DNA damage checkpoints. RESULTS Limited D-J rearrangement patterns distinct from normal thymus were detected at high frequencies (43 of 111 for 40-day thymus and 21 of 45 for 80-day thymus). Those clonally expanded thymocytes mostly consisted of CD4(+)CD8(+) double-positive cells, indicating the retention of lineage capability. They exhibited pausing at a late G1 phase of cell cycle progression but did not show the activation of DNA damage checkpoints such as gammaH2AX, Chk1/2, or p53. Of interest is that 17 of the 52 thymuses showing normal D-J rearrangement patterns at 40 days after irradiation showed allelic loss at the Bcl11b tumor suppressor locus, also indicating clonal expansion. CONCLUSION The thymocytes of clonal growth detected resemble human chronic myeloid leukemia in possessing self-renewal and lineage capability, and therefore they can be a candidate of the lymphoma-initiating cells.

Human T-cell leukemia virus type 1 Tax oncoprotein represses the expression of the BCL11B tumor suppressor in T-cells

Human T‐cell leukemia virus type 1 (HTLV‐1) is the etiological agent of adult T cell leukemia (ATL), which is an aggressive form of T‐cell malignancy. HTLV‐1 oncoproteins, Tax and HBZ, play crucial roles in the immortalization of T‐cells and/or leukemogenesis by dysregulating the cellular functions in the host. Recent studies show that HTLV‐1‐infected T‐cells have reduced expression of the BCL11B tumor suppressor protein. In the present study, we explored whether Tax and/or HBZ play a role in downregulating BCL11B in HTLV‐1‐infected T‐cells. Lentiviral transduction of Tax in a human T‐cell line repressed the expression of BCL11B at both the protein and mRNA levels, whereas the transduction of HBZ had little effect on the expression. Tax mutants with a decreased activity for the NF‐κB, CREB or PDZ protein pathways still showed a reduced expression of the BCL11B protein, thereby implicating a different function of Tax in BCL11B downregulation. In addition, the HTLV‐2 Tax2 protein reduced the BCL11B protein expression in T‐cells. Seven HTLV‐1‐infected T‐cell lines, including three ATL‐derived cell lines, showed reduced BCL11B mRNA and protein expression relative to an uninfected T‐cell line, and the greatest reductions were in the cells expressing Tax. Collectively, these results indicate that Tax is responsible for suppressing BCL11B protein expression in HTLV‐1‐infected T‐cells; Tax‐mediated repression of BCL11B is another mechanism that Tax uses to promote oncogenesis of HTLV‐1‐infected T‐cells.

Successful management of severe intrahepatic cholestasis of pregnancy: report of a first Japanese case

BackgroundIntrahepatic cholestasis of pregnancy (ICP) is a cholestasis condition caused by elevated levels of serum bile acids that mainly occurs in the third trimester of pregnancy. Maternal symptoms include pruritus; elevation of transaminases, biliary enzymes, and bilirubin levels; and abnormal liver function tests. Fetal symptoms include spontaneous preterm labor, fetal distress, and intrauterine death. It is more prevalent in the Caucasians and is rarely found in Asian countries, including Japan. The etiology of ICP has been reported as involving various factors such as, environmental factors, hormone balance, and genetic components. The genetic factors include single-nucleotide polymorphisms (SNPs) in the genes of canalicular transporters, including ABCB4 and ABCB11. It has also been reported that the combination of these SNPs induces severe cholestasis and liver dysfunction.Case presentationHere, we report for the first time a 24-year Japanese case of severe ICP diagnosed by typical symptoms, serum biochemical analysis, and treated with the administration of ursodeoxycholic acid which improved cholestasis and liver injury and prevented fetal death. The sequence analysis showed SNPs reported their association with ICP in the ABCB11 (rs2287622, V444A) and ABCB4 (rs1202283, N168N) loci.ConclusionThe risk of ICP has been reported to be population-specific, and it is rare in the Japanese population. Our case was successfully treated with ursodeoxycholic acid and the genetic sequence analysis has supported the diagnosis. Because genetic variation in ABCB4 and ABCB11 has also been reported in the Japanese population, we need to be aware of potential ICP cases in pregnant Japanese women although further studies are necessary.