Daisaku Miura

N-terminal truncation of Lats1 causes abnormal cell growth control and chromosomal instability.

Summary The tumor suppressors Lats1 and Lats2 are mediators of the Hippo pathway that regulates tissue growth and proliferation. Their N-terminal non-kinase regions are distinct except for Lats conserved domains 1 and 2 (LCD1 and LCD2), which may be important for Lats1/2-specific functions. Lats1 knockout mice were generated by disrupting the N-terminal region containing LCD1 (Lats1&Dgr;N/&Dgr;N). Some Lats1&Dgr;N/&Dgr;N mice were born safely and grew normally. However, mouse embryonic fibroblasts (MEFs) from Lats1&Dgr;N/&Dgr;N mice displayed mitotic defects, centrosomal overduplication, chromosomal misalignment, multipolar spindle formation, chromosomal bridging and cytokinesis failure. They also showed anchorage-independent growth and continued cell cycles and cell growth, bypassing cell-cell contact inhibition similar to tumor cells. Lats1&Dgr;N/&Dgr;N MEFs produced tumors in nude mice after subcutaneous injection, although the tumor growth rate was much slower than that of ordinary cancer cells. Yap, a key transcriptional coactivator of the Hippo pathway, was overexpressed and stably retained in Lats1&Dgr;N/&Dgr;N MEFs in a cell density independent manner, and Lats2 mRNA expression was downregulated. In conclusion, N-terminally truncated Lats1 induced Lats2 downregulation and Yap protein accumulation, leading to chromosomal instability and tumorigenesis.

Cisplatin-induced acute renal failure in mice is mediated by chymase-activated angiotensin-aldosterone system and interleukin-18.

Mechanism(s) of cisplatin-induced acute renal failure, as manifested by increases in blood urea nitrogen and creatinine, was evaluated in relation to production and activation of endogenous mediator(s) in mice. In interleukin (IL)-18-deficient (IL-18KO) mice, cisplatin failed to induce acute renal failure. Administration of recombinant IL-18 prior to cisplatin restored acute renal failure in IL-18KO mice. Accumulation of cisplatin in the kidney was not different in IL-18KO and wild-type (WT) mice, but, clearance of cisplatin was more rapid in IL-18KO mice than in WT mice. Cisplatin increased serum levels of aldosterone and angiotensin II in WT mice, but only angiotensin II levels in IL-18 KO mice. Administration of IL-18 augmented plasma levels of aldosterone and angiotensin II in WT mice. Eplerenone, an aldosterone receptor blocker, TY-51469, a chymase inhibitor and PD123319, a selective angiotensin II type 2 (AT2) receptor antagonist, but not benazepril, an angiotensin-converting enzyme inhibitor, and candesartan, a selective angiotensin II type 1 (AT1) receptor antagonist improved acute renal failure caused by cisplatin, confirming involvement of IL-18, aldosterone and angiotensin II in cisplatin-induced, chymase-dependent acute renal failure in mice. These results show that IL-18, aldosterone and angiotensin II synergistically act to prolong the accumulation of cisplatin in the kidney, leading to acute renal failure. Combined therapy with inhibitors for chymase and aldosterone receptors or AT2 receptors might reduce acute renal failure induced by cisplatin.

Camellia oil and its distillate fractions effectively inhibit the spontaneous metastasis of mouse melanoma BL6 cells

We previously reported that daily intraperitoneal injections of oleamide weakly inhibits the spontaneous metastasis of BL6 cells by blocking the gap junction‐mediated intercellular communications (GJIC) of connexin 26 (Cx26). In the present study, we tested camellia oil, olive oil and cottonseed oil which are rich in oleamide‐like oleic acid for their inhibitory potency on Cx26‐mediated GJIC and spontaneous metastasis of BL6 cells. We found that camellia oil, olive oil and cottonseed oil, and their distillate fractions inhibited Cx26‐mediated GJIC. We also showed that daily intraperitoneal injection of camellia oil and its distillate fractions more potently inhibited spontaneous lung metastasis of BL6 cells than oleamide. Moreover, a daily oral administration of camellia oil distillate fraction effectively inhibited spontaneous metastasis. Notably, even camellia Tempura‐oil, a commercially available food, weakly inhibited the spontaneous metastasis of BL6 cells. Since these oils are used as foods and are quite safe, we propose that they could be used as supplements to protect patients from lung metastasis of melanomas.

Oleamide derivatives suppress the spontaneous metastasis by inhibiting connexin 26

We previously reported that overexpressing connexin 26 (Cx26) enhances the spontaneous metastasis of mouse BL6 melanoma cells. In contrast, daily intraperitoneal injections of an oleamide derivative named MI‐18 potently inhibits the spontaneous metastasis of BL6 cells. In the present study, we chemically synthesized a novel oleamide derivative named MI‐22 and found that it also efficiently suppressed the spontaneous metastasis of BL6 cells. Both MI‐18 and MI‐22 inhibited the gap junction‐mediated intercellular communications (GJIC) that are formed between HeLa cells by the ectopic expression of the hCx26 and hCx32 human connexin subtypes; however, they had no effect on GJIC mediated by hCx40, hCx43 or hCx45. Fluorescently labeled MI‐18 primarily localized not only at plasma membrane but also at Golgi/endosome. This suggests that this oleamide derivative may also act on the Cx26 molecules that accumulate in the Golgi/endosome because of their overexpression. Notably, neither derivative had a cytotoxic effect on HeLa cells when they were added into the tissue culture medium. Taken together, we propose that the MI‐18 and MI‐22 oleamide derivatives may serve as prototypes for novel and clinically important anticancer drugs.

Lats1 suppresses centrosome overduplication by modulating the stability of Cdc25B.

Numerical aberration of the centrosome results in chromosome missegregation, eventually leading to chromosomal instability, a hallmark of human tumor malignancy. Large tumor suppressors 1 and 2 (Lats1 and Lats2) are central kinases in the Hippo pathway and regulate development and tumorigenesis by coordinating the balance between cell proliferation and apoptosis. Importantly, Lats1 and Lats2 also play pivotal roles in cell cycle checkpoint and mitosis. The Lats proteins localize at centrosomes, but their centrosomal functions remain elusive. Here, we generated Lats1-null knockout (Lats1−/−) mice and established Lats1-null mouse embryonic fibroblasts (MEFs). In Lats1−/− MEFs, centrosomes were markedly overduplicated, leading to severe mitotic defects such as chromosome missegregation and cytokinesis failure. We also found that Lats1 physically interacts with Cdc25B phosphatase that localizes both at the centrosome and in the nucleus and regulates the linkage between the centrosome cycle and mitotic progression. Although Lats1 did not phosphorylate Cdc25B, loss of Lats1 in MEFs caused abnormal accumulation of Cdc25B protein and hyperactivation of Cdk2 toward nucleophosmin (NPM/B23), one of the licensing factors involved in centriole duplication. Taken together, these data suggest that Lats1 regulates Cdc25B protein level and subsequent Cdk2 activity, thereby suppressing centrosome overduplication during interphase.

Comprehensive phenotypic analysis of knockout mice deficient in cyclin G1 and cyclin G2

Cyclin G1 (CycG1) and Cyclin G2 (CycG2) play similar roles during the DNA damage response (DDR), but their detailed roles remain elusive. To investigate their distinct roles, we generated knockout mice deficient in CycG1 (G1KO) or CycG2 (G2KO), as well as double knockout mice (DKO) deficient in both proteins. All knockouts developed normally and were fertile. Generation of mouse embryonic fibroblasts (MEFs) from these mice revealed that G2KO MEFs, but not G1KO or DKO MEFs, were resistant to DNA damage insults caused by camptothecin and ionizing radiation (IR) and underwent cell cycle arrest. CycG2, but not CycG1, co-localized with γH2AX foci in the nucleus after γ-IR, and γH2AX-mediated DNA repair and dephosphorylation of CHK2 were delayed in G2KO MEFs. H2AX associated with CycG1, CycG2, and protein phosphatase 2A (PP2A), suggesting that γH2AX affects the function of PP2A via direct interaction with its B’γ subunit. Furthermore, expression of CycG2, but not CycG1, was abnormal in various cancer cell lines. Kaplan–Meier curves based on TCGA data disclosed that head and neck cancer patients with reduced CycG2 expression have poorer clinical prognoses. Taken together, our data suggest that reduced CycG2 expression could be useful as a novel prognostic marker of cancer.

Isolation of cancer cells with augmented spheroid-forming capability using a novel tool equipped with removable filter

Three-dimensional (3D) cell culture systems have been used to obtain multicellular spheroidal cell aggregates, or spheroids, from cancer cells. However, it is difficult to efficiently prepare large tumor-derived spheroids from cancer cells. To circumvent this problem, we here used a tool equipped with removal membrane, called Spheroid Catch, for the selection and enrichment of large-sized and/or size-matched spheroids from human squamous cell carcinoma (SAS cells) without loss of recovery. After a five-round process of selection and enrichment, we successfully isolated a subpopulation of SAS cells with augmented spheroid-forming capability, named eSAS: the efficiency of spheroid formation is 28.5% (eSAS) vs 16.8% (parental SAS). Notably, we found that some of eSAS cells survived after exposure of high doses of cisplatin in 3D culture. Moreover, orthotopic implantation by injecting eSAS cells into the tongues of nude mice showed reduced survival rate and increased tumor growth compared with those of nude mice injected with SAS cells. These results suggest that spheroids exhibiting properties of higher spheroid forming capacity can be efficiently collected by using Spheroid Catch. Indeed, genome-wide cDNA microarray and western blot analyses demonstrated higher mRNA and protein levels of hedgehog acyltransferase (HHAT), which is associated with stem maintenance in cell carcinoma by catalysing the N-palmitoylation of Hedgehog proteins, in eSAS cells than in SAS cells. We propose that Spheroid Catch could be useful for the study of spheroids, and potentially organoids, in the basic and clinical sciences, as an alternative method to other type of cell strainers.