The CD44/COL17A1 pathway promotes the formation of multilayered, transformed epithelia.

At the early stage of cancer development, oncogenic mutations often cause multilayered epithelial structures. However, the underlying molecular mechanism still remains enigmatic. By performing a series of screenings targeting plasma membrane proteins, we have found that collagen XVII (COL17A1) and CD44 accumulate in RasV12-, Src-, or ErbB2-transformed epithelial cells. In addition, the expression of COL17A1 and CD44 is also regulated by cell density and upon apical cell extrusion. We further demonstrate that the expression of COL17A1 and CD44 is profoundly upregulated at the upper layers of multilayered, transformed epithelia in vitro and in vivo. The accumulated COL17A1 and CD44 suppress mitochondrial membrane potential and reactive oxygen species (ROS) production. The diminished intracellular ROS level then promotes resistance against ferroptosis-mediated cell death upon cell extrusion, thereby positively regulating the formation of multilayered structures. To further understand the functional role of COL17A1, we performed comprehensive metabolome analysis and compared intracellular metabolites between RasV12 and COL17A1-knockout RasV12 cells. The data imply that COL17A1 regulates the metabolic pathway from the GABA shunt to mitochondrial complex I through succinate, thereby suppressing the ROS production. Moreover, we demonstrate that CD44 regulates membrane accumulation of COL17A1 in multilayered structures. These results suggest that CD44 and COL17A1 are crucial regulators for the clonal expansion of transformed cells within multilayered epithelia, thus being potential targets for early diagnosis and preventive treatment for precancerous lesions.

LY6D-induced macropinocytosis as a survival mechanism of senescent cells.

Although senescent cells display various morphological changes including vacuole formation, it is still unclear how these processes are regulated. We have recently identified the gene, lymphocyte antigen 6 complex, locus D (LY6D), to be upregulated specifically in senescent cells. LY6D is a glycosylphosphatidylinositol-anchored cell-surface protein whose function remains unknown. Here, we analyzed the functional relationship between LY6D and the senescence processes. We found that overexpression of LY6D induced vacuole formation and knockdown of LY6D suppressed the senescence-associated vacuole formation. The LY6D-induced vacuoles were derived from macropinocytosis, a distinct form of endocytosis. Furthermore, Src family kinases and Ras were found to be recruited to membrane lipid rafts in an LY6D-dependent manner, and inhibition of their activity impaired the LY6D-induced macropinocytosis. Finally, reduction of senescent-cell survival induced by glutamine deprivation was recovered by albumin supplementation to the culture media in an LY6D-dependent manner. Because macropinocytosis acts as an amino acid supply route, these results suggest that LY6D-mediated macropinocytosis contributes to senescent-cell survival through the incorporation of extracellular nutrients.

Identification of natural rubber degradation gene in Rhizobacter gummiphilus NS21.

A Gram-negative rubber-degrading bacterium, Rhizobacter gummiphilus NS21 grew and produced aldehyde metabolites on a deproteinized natural rubber (DPNR)-overlay agar medium forming a clearing zone. A transposon-insertion mutant, which had lost the ability to degrade DPNR, was isolated to identify the rubber degradation genes. Sequencing analysis indicated that the transposon was inserted into a putative oxygenase gene, latA. The deduced amino acid sequence of latA has 36% identity with that of roxA, which encodes a rubber oxygenase of Xanthomonas sp. strain 35Y. Phylogenetic analysis revealed that LatA constitutes a distinct group from RoxA. Heterologous expression in a Methylibium host and deletion analysis of latA indicated that the latA product is responsible for the depolymerization of DPNR. The quantitative reverse transcription-PCR analysis indicated that the transcription of latA is induced during the growth on DPNR. These results strongly suggest that latA is directly involved in the degradation of rubber in NS21.