Takashi Nishizawa

Recurrent gain-of-function mutations of RHOA in diffuse-type gastric carcinoma

Diffuse-type gastric carcinoma (DGC) is characterized by a highly malignant phenotype with prominent infiltration and stromal induction. We performed whole-exome sequencing on 30 DGC cases and found recurrent RHOA nonsynonymous mutations. With validation sequencing of an additional 57 cases, RHOA mutation was observed in 25.3% (22/87) of DGCs, with mutational hotspots affecting the Tyr42, Arg5 and Gly17 residues in RHOA protein. These positions are highly conserved among RHO family members, and Tyr42 and Arg5 are located outside the guanine nucleotide–binding pocket. Several lines of functional evidence indicated that mutant RHOA works in a gain-of-function manner. Comparison of mutational profiles for the major gastric cancer subtypes showed that RHOA mutations occur specifically in DGCs, the majority of which were histopathologically characterized by the presence of poorly differentiated adenocarcinomas together with more differentiated components in the gastric mucosa. Our findings identify a potential therapeutic target for this poor-prognosis subtype of gastric cancer with no available molecularly targeted drugs.

The effect of paclobutrazol on growth and yield during first year greenhouse strawberry production

Abstract On 25 June 1991, PP333 was applied as a soil treatment to strawberry plants cultivar ‘Nyoho’ at the rates of 0, 0.01, 0.04, 0.09, 0.16 and 0.25 mg a.i. per pot. On 12 August, the plants were transferred to a growth chamber maintained at 17 14 ° C under 11-h photoperiod and grown for 21 days in order to induce flower buds. On 28 September, the plants were transferred to a glasshouse and the ripe berries were harvested at 1 to 2 day intervals until 31 December. As the concentrations of PP333 increased, the length and number of runners decreased; however, the numbers of lateral crowns and flower clusters increased. PP333 reduced the petiole length and size of leaves which unfolded prior to the harvesting season of the berries. However, the growth of the leaves was back to normal by 31 December, about 6 months after the treatment had started. When PP333 was applied at rates of 0.04–0.09 mg a.i. per pot, the dry weights of both roots and crowns (the initial harvesting time of berries) had become higher than those of the untreated plants by 14 November. However, their dry weights on 31 December had become lower than those of the untreated plants. In the first year growing period, the plants that had PP333 applied at rates of 0.04–0.16 mg a.i. per pot had an increased number of ripe berries and an increased berry yield. The ripening of the berries, however, was delayed by about 5 days.

DGC-specific RHOA mutations maintained cancer cell survival and promoted cell migration via ROCK inactivation

RHOA missense mutations exist specifically in diffuse type gastric cancers (DGC) and are considered one of the DGC driver genes, but it is not fully understood how RHOA mutations contribute to DGC development. Here we examined how RHOA mutations affect cancer cell survival and cell motility. We revealed that cell survival was maintained by specific mutation sites, namely G17, Y42, and L57. Because these functional mutations suppressed MLC2 phosphorylation and actin stress fiber formation, we realized they act in a dominant-negative fashion against the ROCK pathway. Through the same inactivating mechanism that maintained cell survival, RHOA mutations also increased cell migration activity. Cell survival and migration studies on CLDN18-ARHGAP (CLG) fusions, which are known to be mutually exclusive to RHOA mutations, showed that CLG fusions complemented cell survival under RHOA knockdown condition and also induced cell migration. Site-directed mutagenesis analysis revealed the importance of the GAP domain and indicated that CLG fusions maintained RHOA in the inactive form. Taken together, these findings show that the inactivation of ROCK would be a key step in DGC development, so ROCK activation might provide novel therapeutic opportunities.

Difference in morphology and interactome profiles between orthotopic and subcutaneous gastric cancer xenograft models

In xenograft models, orthotopic (ORT) engraftment is thought to provide a different tumor microenvironment compared with subcutaneous (SC) engraftment. We attempted to characterize the biological difference between OE19 (adenocarcinoma of the gastroesophageal junction) SC and ORT models by pathological analysis and CASTIN (CAncer-STromal INteractome) analysis, which is a novel method developed to analyze the tumor-stroma interactome framework. In SC models, SCID mice were inoculated subcutaneously with OE19 cells, and tumor tissues were sampled at 3 weeks. In ORT models, SCID mice were inoculated under the serosal membrane of the stomach wall, and tumor tissues were sampled at 3 and 6 weeks after engraftment. Results from the two models were then compared. Histopathologically, the SC tumors were well circumscribed from the adjacent tissue, with scant stroma and the formation of large ductal structures. In contrast, the ORT tumors were less circumscribed, with small ductal structures invading into abundant stroma. Then we compared the transcriptome profiles of human tumor cells with the mouse stromal cells of each model by species-specific RNA sequencing. With CASTIN analysis, we successfully identified several interactions that are known to affect the tumor microenvironment as being selectively enhanced in the ORT model. In conclusion, pathological analysis and CASTIN analysis revealed that ORT models of OE19 cells have a more invasive character and enhanced interaction with stromal cells compared with SC models.