Noritaka Yamaguchi

NOTCH3 Signaling Pathway Plays Crucial Roles in the Proliferation of ErbB2-Negative Human Breast Cancer Cells

ErbB2-negative breast tumors represent a significant therapeutic hurdle because of a lack of effective molecular targets. Although NOTCH proteins are known to be involved in mammary tumorigenesis, the functional significance of these proteins in ErbB2-negative breast tumors is not clear. In the present study, we examined the expression of activated NOTCH receptors in human breast cancer cell lines, including ErbB2-negative and ErbB2-positive cell lines. Activated NOTCH1 and NOTCH3 proteins generated by gamma-secretase were detected in most of the cell lines tested, and both proteins activated CSL-mediated transcription. Down-regulation of NOTCH1 by RNA interference had little or no suppressive effect on the proliferation of either ErbB2-positive or ErbB2-negative cell lines. In contrast, down-regulation of NOTCH3 significantly suppressed proliferation and promoted apoptosis of the ErbB2-negative tumor cell lines. Down-regulation of NOTCH3 did not have a significant effect on the ErbB2-positive tumor cell lines. Down-regulation of CSL also suppressed the proliferation of ErbB2-negative breast tumor cell lines, indicating that the NOTCH-CSL signaling axis is involved in cell proliferation. Finally, NOTCH3 gene amplification was detected in a breast tumor cell line and one breast cancer tissue specimen even though the frequency of NOTCH3 gene amplification was low (<1%). Taken together, these findings indicate that NOTCH3-mediated signaling rather than NOTCH1-mediated signaling plays an important role in the proliferation of ErbB2-negative breast tumor cells and that targeted suppression of this signaling pathway may be a promising strategy for the treatment of ErbB2-negative breast cancers.

NF-κB non-cell-autonomously regulates cancer stem cell populations in the basal-like breast cancer subtype

Patients with triple-negative breast cancer display the highest rates of early relapse of all patients with breast cancer. The basal-like subtype, a subgroup of triple-negative breast cancer, exhibits high levels of constitutively active NF-κB signalling. Here we show that NF-κB activation, induced by inflammatory cytokines or by epigenetically dysregulated NIK expression, cell-autonomously upregulates JAG1 expression in non-cancer stem cells. This upregulation stimulates NOTCH signalling in cancer stem cells in trans, leading to an expansion of cancer stem cell populations. Among breast cancers, the NF-κB-dependent induction of JAG1 and the NOTCH-dependent expansion of the cancer stem cell population occur only in the basal-like subtype. Collectively, our results indicate that NF-κB has a non-cell-autonomous role in regulating cancer stem cell populations by forming intratumoural microenvironments composed of JAG1-expressing non-cancer stem cells with a basal-like subtype.

Constitutive activation of nuclear factor‐κB is preferentially involved in the proliferation of basal‐like subtype breast cancer cell lines

Constitutive nuclear factor (NF)‐κB activation is thought to be involved in survival, invasion, and metastasis in various types of cancers. However, neither the subtypes of breast cancer cells with constitutive NF‐κB activation nor the molecular mechanisms leading to its constitutive activation have been clearly defined. Here, we quantitatively analyzed basal NF‐κB activity in 35 human breast cancer cell lines and found that most of the cell lines with high constitutive NF‐κB activation were categorized in the estrogen receptor negative, progesterone receptor negative, ERBB2 negative basal‐like subtype, which is the most malignant form of breast cancer. Inhibition of constitutive NF‐κB activation by expression of IκBα super‐repressor reduced proliferation of the basal‐like subtype cell lines. Expression levels of mRNA encoding NF‐κB‐inducing kinase (NIK) were elevated in several breast cancer cell lines, and RNA interference‐mediated knockdown of NIK reduced NF‐κB activation in a subset of the basal‐like subtype cell lines with upregulated NIK expression. Taken together, these results suggest that constitutive NF‐κB activation, partially dependent on NIK, is preferentially involved in proliferation of basal‐like subtype breast cancer cells and may be a useful therapeutic target for this subtype of cancer. (Cancer Sci 2009; 100: 1668–1674)

Targeted Disruption of Intracellular Type I Platelet Activating Factor-acetylhydrolase Catalytic Subunits Causes Severe Impairment in Spermatogenesis

Intracellular type I platelet activating factor-acetylhydrolase is a phospholipase that consists of a dimer of two homologous catalytic subunits α1 and α2 as well as LIS1, a product of the causative gene for type I lissencephaly. LIS1 plays an important role in neuronal migration during brain development, but thein vivo function of the catalytic subunits remains unclear. In this study, we generated α1- anda2-deficient mice by targeted disruption.α1−/− mice are indistinguishable from wild-type mice, whereas α2−/− male mice show a significant reduction in testis size. Double-mutant male mice are sterile because of severe impairment of spermatogenesis. Histological examination revealed marked degeneration at the spermatocyte stage and an increase of apoptotic cells in the seminiferous tubules. The catalytic subunits are expressed at high levels in testis as well as brain in mice. In wild-type mice, α2 is expressed in all seminiferous tubule cell types, whereas α1 is expressed only in the spermatogonia. This expression pattern parallels the finding that deletion of both subunits induces a marked loss of germ cells at an early spermatogenic stage. We also found that the LIS1 protein levels, but not the mRNA levels, were significantly reduced in α2−/− and double-mutant mice, suggesting that the catalytic subunits, especially α2, are a determinant of LIS1 expression level.

Involvement of A20 in the molecular switch that activates the non-canonical NF-кB pathway

The non-canonical NF-κB pathway is crucial for the immune system. A critical event in activation of the non-canonical pathway is the attenuation of NF-κB-inducing kinase (NIK) degradation, which is promoted by continuous polyubiquitination of NIK catalyzed by the NIK ubiquitin-ligase complex composed of cellular inhibitor of apoptosis protein 1 and 2 (cIAP1/2), TNF receptor-associated factor 2 (TRAF2), and TRAF3. However, the molecular mechanism of stimulation-dependent NIK stabilization remains poorly understood. Here, we show that A20, a ubiquitin-editing enzyme, promotes efficient activation of the non-canonical pathway independent of its catalytic activity. A20 directly binds to cIAP1 through the seventh zinc finger of A20, resulting in dissociation of the TRAF2/TRAF3 interaction, thereby inactivating the ligase complex to stabilize NIK. Given that A20 negatively regulates the canonical pathway, A20 is likely involved in the molecular switch that promotes the transition from canonical to non-canonical activation for proper control of the immune system.

Phosphorylation of KRAB-associated Protein 1 (KAP1) at Tyr-449, Tyr-458, and Tyr-517 by Nuclear Tyrosine Kinases Inhibits the Association of KAP1 and Heterochromatin Protein 1α (HP1α) with Heterochromatin

Background: We showed that nuclear tyrosine phosphorylation is involved in chromatin structural changes. Results: Several tyrosine kinases phosphorylate KAP1 at Tyr-449, Tyr-458, and Tyr-517 in the nucleus, resulting in a decrease of KAP1 association with heterochromatin. Conclusion: Tyrosine phosphorylation of KAP1 by nucleus-localized tyrosine kinases, including Src, involves heterochromatin structural changes. Significance: These findings provide a new insight into nuclear tyrosine phosphorylation signals. Protein tyrosine phosphorylation regulates a wide range of cellular processes at the plasma membrane. Recently, we showed that nuclear tyrosine phosphorylation by Src family kinases (SFKs) induces chromatin structural changes. In this study, we identify KRAB-associated protein 1 (KAP1/TIF1β/TRIM28), a component of heterochromatin, as a nuclear tyrosine-phosphorylated protein. Tyrosine phosphorylation of KAP1 is induced by several tyrosine kinases, such as Src, Lyn, Abl, and Brk. Among SFKs, Src strongly induces tyrosine phosphorylation of KAP1. Nucleus-targeted Lyn potentiates tyrosine phosphorylation of KAP1 compared with intact Lyn, but neither intact Fyn nor nucleus-targeted Fyn phosphorylates KAP1. Substitution of the three tyrosine residues Tyr-449/Tyr-458/Tyr-517, located close to the HP1 binding-motif, into phenylalanine ablates tyrosine phosphorylation of KAP1. Immunostaining and chromatin fractionation show that Src and Lyn decrease the association of KAP1 with heterochromatin in a kinase activity-dependent manner. KAP1 knockdown impairs the association of HP1α with heterochromatin, because HP1α associates with KAP1 in heterochromatin. Intriguingly, tyrosine phosphorylation of KAP1 decreases the association of HP1α with heterochromatin, which is inhibited by replacement of endogenous KAP1 with its phenylalanine mutant (KAP1-Y449F/Y458F/Y517F, KAP1–3YF). In DNA damage, KAP1–3YF repressed transcription of p21. These results suggest that nucleus-localized tyrosine kinases, including SFKs, phosphorylate KAP1 at Tyr-449/Tyr-458/Tyr-517 and inhibit the association of KAP1 and HP1α with heterochromatin.

NIK is involved in constitutive activation of the alternative NF-κB pathway and proliferation of pancreatic cancer cells

Pancreatic cancer has one of the poorest prognoses among human neoplasms. Constitutive activation of NF-kappaB is frequently observed in pancreatic cancer cells and is involved in their malignancy. However, little is known about the molecular mechanism of this constitutive NF-kappaB activation. Here, we show that the alternative pathway is constitutively activated and NF-kappaB-inducing kinase (NIK), a mediator of the alternative pathway, is significantly expressed in pancreatic cancer cells. siRNA-mediated silencing of NIK expression followed by subcellular fractionation revealed that NIK is constitutively involved in the processing of p100 and nuclear transport of p52 and RelB in pancreatic cancer cells. In addition, NIK silencing significantly suppressed proliferation of pancreatic cancer cells. These results clearly indicate that NIK is involved in the constitutive activation of the alternative pathway and controls cell proliferation in pancreatic cancer cells. Therefore, NIK might be a novel target for the treatment of pancreatic cancer.

Activation of the Prereplication Complex Is Blocked by Mimosine through Reactive Oxygen Species-activated Ataxia Telangiectasia Mutated (ATM) Protein without DNA Damage

Background: Mimosine is a cell synchronization reagent used for arresting cells in late G1 and S phases. Results: Replication fork assembly is reversibly blocked by ATM activation through mimosine-generated reactive oxygen species. Conclusion: Mimosine induces cell cycle arrest strictly at the G1-S phase boundary, which prevents replication fork stalling-induced DNA damage. Significance: These findings provide a novel mechanism of the mimosine-induced G1 checkpoint. Mimosine is an effective cell synchronization reagent used for arresting cells in late G1 phase. However, the mechanism underlying mimosine-induced G1 cell cycle arrest remains unclear. Using highly synchronous cell populations, we show here that mimosine blocks S phase entry through ATM activation. HeLa S3 cells are exposed to thymidine for 15 h, released for 9 h by washing out the thymidine, and subsequently treated with 1 mm mimosine for a further 15 h (thymidine → mimosine). In contrast to thymidine-induced S phase arrest, mimosine treatment synchronizes >90% of cells at the G1-S phase boundary by inhibiting the transition of the prereplication complex to the preinitiation complex. Mimosine treatment activates ataxia telangiectasia mutated (ATM)/ataxia telangiectasia and Rad3-related (ATR)-mediated checkpoint signaling without inducing DNA damage. Inhibition of ATM activity is found to induce mimosine-arrested cells to enter S phase. In addition, ATM activation by mimosine treatment is mediated by reactive oxygen species (ROS). These results suggest that, upon mimosine treatment, ATM blocks S phase entry in response to ROS, which prevents replication fork stalling-induced DNA damage.

Epigenetic alteration of the NF‐κB‐inducing kinase (NIK) gene is involved in enhanced NIK expression in basal‐like breast cancer

Basal‐like breast cancers are triple‐negative (estrogen receptor negative, progesterone receptor negative, erythroblastic leukemia viral oncogene homolog 2 (ERBB2) negative) tumors with an aggressive clinical behavior that lacks effective molecular targets for therapy. We reported previously that the basal‐like subtype cell lines display high constitutive nuclear factor (NF)‐κB activation, whose inhibition in the basal‐like subtypes suppressed their proliferation. Moreover, NF‐κB‐inducing kinase (NIK) is involved in the constitutive NF‐κB activation. Here, we report that enhanced NIK expression, which is exclusively observed in the basal‐like subtype rather than the luminal‐like subtype or non‐tumorigenic mammary epithelial cells, is caused by epigenetic alteration of the NIK gene. The stability of NIK mRNA and transcriptional activity driven by the NIK promoter are similar in the basal‐like and luminal‐like subtypes. However, histone H3 acetylation levels were up‐regulated in the basal‐like subtype. Furthermore, treatment of the luminal‐like subtype with a histone deacetylase inhibitor, valproic acid, significantly increased NIK expression. Although DNA methylation of the NIK locus was not detected, NIK expression also increased when the luminal‐like subtype was treated with 5‐azacytidine, which inhibits histone H3‐Lys‐9 dimethylation in addition to DNA methylation. Taken together, these results suggest that the closed chromatin structure mediated by histone H3 methylation and deacetylation suppresses NIK expression in the luminal‐like subtype, whereas disruption of these suppression mechanisms leads to enhanced NIK expression and the constitutive NF‐κB activation in the basal‐like subtype. Thus, NIK and genes induced by the NIK‐mediated constitutive NF‐κB activation could be therapeutic targets of basal‐like breast cancer. (Cancer Sci 2010; 101: 2391–2397)

Formation of long and winding nuclear F-actin bundles by nuclear c-Abl tyrosine kinase

The non-receptor-type tyrosine kinase c-Abl is involved in actin dynamics in the cytoplasm. Having three nuclear localization signals (NLSs) and one nuclear export signal, c-Abl shuttles between the nucleus and the cytoplasm. Although monomeric actin and filamentous actin (F-actin) are present in the nucleus, little is known about the relationship between c-Abl and nuclear actin dynamics. Here, we show that nuclear-localized c-Abl induces nuclear F-actin formation. Adriamycin-induced DNA damage together with leptomycin B treatment accumulates c-Abl into the nucleus and increases the levels of nuclear F-actin. Treatment of c-Abl-knockdown cells with Adriamycin and leptomycin B barely increases the nuclear F-actin levels. Expression of nuclear-targeted c-Abl (NLS-c-Abl) increases the levels of nuclear F-actin even without Adriamycin, and the increased levels of nuclear F-actin are not inhibited by inactivation of Abl kinase activity. Intriguingly, expression of NLS-c-Abl induces the formation of long and winding bundles of F-actin within the nucleus in a c-Abl kinase activity-dependent manner. Furthermore, NLS-c-AblΔC, which lacks the actin-binding domain but has the full tyrosine kinase activity, is incapable of forming nuclear F-actin and in particular long and winding nuclear F-actin bundles. These results suggest that nuclear c-Abl plays critical roles in actin dynamics within the nucleus.