Terminal differentiation is an important event for maintaining normal homeostasis in the colorectal epithelium, and the loss of apoptosis is an important mechanism underlying colorectal tumorigenesis. The very limited current data on the role of lipoxygenase (LOX) metabolism in tumorigenesis suggests that the oxidative metabolism of linoleic and arachidonic acid possibly shifts from producing antitumorigenic 15-LOX-1 and 15-LOX-2 products to producing protumorigenic 5-LOX and 12-LOX products. We examined whether this shift occurs in vitro in the human colon cancer cell line Caco-2 in association with the loss of terminal differentiation and apoptosis, or in vivo during the formation of colorectal adenomas in patients with familial adenomatous polyposis (FAP). Restoring terminal differentiation and apoptosis of Caco-2 cells increased the mRNA levels of 5-LOX, 15-LOX-2, and 15-LOX-1, but the only significant increases in protein expression and enzymatic activity were of 15-LOX-1. In FAP patients, 15-LOX-1 expression and activity were significantly down-regulated in adenomas (compared with paired nonneoplastic epithelial mucosa), whereas 5-LOX and 15-LOX-2 protein expressions and enzymatic activities were not. We conducted a validation study with immunohistochemical testing in a second group of FAP patients; 15-LOX-1 expression was down-regulated in colorectal adenomas (compared with nonneoplastic epithelial mucosa) in 87% (13 of 15) of this group. We confirmed the mechanistic relevance of these findings by demonstrating that ectopically restoring 15-LOX-1 expression reestablished apoptosis in Caco-2 cells. Therefore, 15-LOX-1 down-regulation rather than a shift in the balance of LOXs is likely the dominant alteration in LOX metabolism which contributes to colorectal tumorigenesis by repressing apoptosis. (Cancer Res 2005; 65(24): 11486-92)

Expression of 15-lipoxygenase-1 (15-LOX-1) is decreased in many human cancers; however, the mechanistic significance of its decreased expression has been difficult to determine because its mouse homolog 12/15-LOX has opposing functions. We generated a mouse model in which expression of a human 15-LOX-1 transgene was targeted to the intestinal epithelium via the villin promoter. Targeted expression was confirmed by real-time reverse transcription-polymerase chain reaction and immunoblotting. When the 15-LOX-1 transgene was expressed in colonic epithelial cells of two independent mouse lines (B6 and FVB), azoxymethane-inducible colonic tumorigenesis was suppressed (mean number of tumors: wild type [WT] = 8.2, 15-LOX-1(+/-) = 4.91, 15-LOX-1(+/+) = 3.57; WT vs 15-LOX-1(+/-) two-sided P = .003, WT vs 15-LOX-1(+/+) two-sided P < .001; n = 10-14 mice per group). 15-LOX-1 transgene expression was always decreased in the tumors that did develop. In the presence of expression of the 15-LOX-1 transgene, expression of tumor necrosis factor alpha and its target inducible nitric oxide synthase were decreased and activation of nuclear factor-kappa B in colonic epithelial cells was inhibited.

Loss of terminal cell differentiation promotes tumorigenesis. 15-LOX-1 contributes to terminal cell differentiation in normal cells. The mechanistic significance of 15-LOX-1 expression loss in human cancers to terminal cell differentiation suppression is unknown. In a screen of 128 cancer cell lines representing more than 20 types of human cancer, we found that 15-LOX-1 mRNA expression levels were markedly lower than levels in terminally differentiated cells. Relative expression levels of 15-LOX-1 (relative to the level in terminally differentiated primary normal human-derived bronchial epithelial cells) were lower in 79% of the screened cancer cell lines than relative expression levels of p16 (INK4A), which promotes terminal cell differentiation and is considered one of the most commonly lost tumor suppressor genes in cancer cells. 15-LOX-1 was expressed during terminal differentiation in three-dimensional air-liquid interface cultures, and 15-LOX-1 expression and terminal differentiation occurred in immortalized non-transformed bronchial epithelial but not lung cancer cell lines. 15-LOX-1 expression levels were lower in human tumors than paired normal lung epithelia. Short hairpin RNA-mediated downregulation of 15-LOX-1 in Caco-2 cells blocked enterocyte-like differentiation, disrupted tight junction formation, and blocked E-cadherin and ZO-1 localization to the cell wall membrane. 15-LOX-1 episomal expression in Caco-2 and HT-29 colon cancer cells induced differentiation. Our findings indicate that 15-LOX-1 downregulation in cancer cells is an important mechanism for terminal cell differentiation dysregulation and support the potential therapeutic utility of 15-LOX-1 re-expression to inhibit tumorigenesis.

The molecular mechanism underlying sex determination and gonadal differentiation of the mud crab (Scylla paramamosain) has received considerable attention, due to the remarkably biological and economic differences between sexes. However, sex-biased genes, especially non-coding genes, which account for these differences, remain elusive in this crustacean species. In this study, the first de novo gonad transcriptome sequencing was performed to identify both differentially expressed genes and long non-coding RNAs (lncRNAs) between male and female S. paramamosain by using Illumina Hiseq2500. A total of 79,282,758 and 79,854,234 reads were generated from ovarian and testicular cDNA libraries, respectively. After filtrating and de novo assembly, 262,688 unigenes were produced from both libraries. Of these unigenes, 41,125 were annotated with known protein sequences in public databases. Homologous genes involved in sex determination and gonadal development pathways (Sxl-Tra/Tra-2-Dsx/Fru, Wnt4, thyroid hormone synthesis pathway, etc.) were identified. Three hundred and sixteen differentially expressed unigenes were further identified between both transcriptomes. Meanwhile, a total of 233,078 putative lncRNAs were predicted. Of these lncRNAs, 147 were differentially expressed between sexes. qRT-PCR results showed that nine lncRNAs negatively regulated the expression of eight genes, suggesting a potential role in sex differentiation. These findings will provide fundamental resources for further investigation on sex differentiation and regulatory mechanism in crustaceans.