Xiangsheng Zuo

The 15-lipoxygenase-1 product 13-S-hydroxyoctadecadienoic acid down-regulates PPAR-δ to induce apoptosis in colorectal cancer cells

Diminished apoptosis, a critical event in tumorigenesis, is linked to down-regulated 15-lipoxygenase-1 (15-LOX-1) expression in colorectal cancer cells. 13-S-hydroxyoctadecadienoic acid (13-S-HODE), which is the primary product of 15-LOX-1 metabolism of linoleic acid, restores apoptosis. Nonsteroidal antiinflammatory drugs (NSAIDs) transcriptionally up-regulate 15-LOX-1 expression to induce apoptosis. Peroxisome proliferator-activated receptors (PPARs) are nuclear receptors for linoleic and arachidonic acid metabolites. PPAR-δ promotes colonic tumorigenesis. NSAIDs suppress PPAR-δ activity in colon cancer cells. The mechanistic relationship between 15-LOX-1 and PPAR-δ was previously unknown. Our current study shows that (i) 13-S-HODE binds to PPAR-δ, decreases PPAR-δ activation, and down-regulates PPAR-δ expression in colorectal cancer cells; (ii) the induction of 15-LOX-1 expression is a critical step in NSAID down-regulation of PPAR-δ and the resultant induction of apoptosis; and (iii) PPAR-δ is an important signaling receptor for 13-S-HODE-induced apoptosis. The in vivo relevance of these mechanistic findings was demonstrated in our tumorigenesis studies in nude mouse xenograft models. Our findings indicate that the down-regulation of PPAR-δ by 15-LOX-1 through 13-S-HODE is an apoptotic signaling pathway that is activated by NSAIDs.

Concise Review: Emerging Role of CD44 in Cancer Stem Cells: A Promising Biomarker and Therapeutic Target

The reception and integration of the plethora of signals a cell receives from its microenvironment determines the cells fate. CD44 functions as a receptor for hyaluronan and many other extracellular matrix components, as well as a cofactor for growth factors and cytokines, and thus, CD44 is a signaling platform that integrates cellular microenvironmental cues with growth factor and cytokine signals and transduces signals to membrane‐associated cytoskeletal proteins or to the nucleus to regulate a variety of gene expression levels related to cell‐matrix adhesion, cell migration, proliferation, differentiation, and survival. Accumulating evidence indicates that CD44, especially CD44v isoforms, are cancer stem cell (CSC) markers and critical players in regulating the properties of CSCs, including self‐renewal, tumor initiation, metastasis, and chemoradioresistance. Furthermore, there is ample evidence that CD44, especially CD44v isoforms, are valuable prognostic markers in various types of tumors. Therefore, therapies that target CD44 may destroy the CSC population, and this holds great promise for the cure of life‐threatening cancers. However, many challenges remain to determining how best to use CD44 as a biomarker and therapeutic target. Here we summarize the current findings concerning the critical role of CD44/CD44v in the regulation of cancer stemness and the research status of CD44/CD44v as biomarkers and therapeutic targets in cancer. We also discuss the current challenges and future directions that may lead to the best use of CD44/CD44v for clinical applications.

Oxidative metabolism of linoleic acid modulates PPAR-beta/delta suppression of PPAR-gamma activity

Peroxisome proliferator-activated receptors (PPARs) are transcription factors that strongly influence molecular events in normal and cancer cells. PPAR-beta/delta (PPAR-b/d) overexpression suppresses the activity of PPAR-gamma (PPAR-g) and PPAR-alpha. This interaction has been questioned, however, by studies with synthetic ligands of PPARs in PPAR-b/d-null cells, and it is not known whether an interaction between PPAR-b/d and PPAR-g exists, especially in relation to the signaling by natural PPAR ligands. Oxidative metabolites of linoleic and arachidonic acids are natural ligands of PPARs. 13-S-hydroxyoctadecadienoic acid (13-S-HODE), the main product of 15-lipoxygenase-1 (15-LOX-1) metabolism of linoleic acid, downregulates PPAR-b/d. We tested (a) whether PPAR-b/d expression modulates PPAR-g activity in experimental models of the loss and gain of PPAR-b/d function in colon cancer cells and (b) whether 15-LOX-1 formation of 13-S-HODE influences the interaction between PPAR-b/d and PPAR-g. We found that (a) 15-LOX-1 formation of 13-S-HODE promoted PPAR-g activity, (b) PPAR-b/d expression suppressed PPAR-g activity in models of both loss and gain of PPAR-b/d function, (c) 15-LOX-1 activated PPAR-g by downregulating PPAR-b/d, and (d) 15-LOX-1 expression induced apoptosis in colon cancer cells via modulating PPAR-b/d suppression of PPAR-g. These findings elucidate a novel mechanism of the signaling by natural ligands of PPARs, which involves modulating the interaction between PPAR-b/d and PPAR-g.

The transcription factor GATA-6 is overexpressed in vivo and contributes to silencing 15-LOX-1 in vitro in human colon cancer

Transcriptional suppression of 15‐li‐poxygenase (LOX)‐1 (15‐LOX‐1) helps enable human colorectal cancer cells escape apoptosis, a critical mechanism for colonic tumorigenesis. GATA‐6 is strongly expressed in vitro in cancer cells;its down‐regulation by pharmaceuticals is associated with reversal of 15‐LOX‐1 transcriptional suppression. The mechanistic contribution of GATA‐6 overexpression to colonic tumorigenesis, especially concerning 15‐LOX‐1 transcriptional suppression, remains unknown. We tested whether GATA‐6 is differentially overexpressed in human colorectal cancers and whether reversing GATA‐6 overexpression in colon cancer cells is sufficient to restore 15‐LOX‐1 expression and influence cell proliferation or apoptosis. The expression of GATA‐6 RNA and protein was measured in paired human colorectal cancer and normal tissues from two separate patient groups. We used GATA‐6 small interfering RNA transfection to down‐regulate GATA‐6 expression and examine the effects of this down‐regulation on 15‐LOX‐1 expression, cell proliferation, and apoptosis in Caco‐2 and HCT‐116 colon cancer cells with and without the nonsteroidal antiinflammatory drug NS‐398 or the histone deacetylase inhibitor sodium butyrate. GATA‐6 mRNA and protein expressions were higher in cancer than normal epithelia of the colon. GATA‐6 knockdown was insufficient by itself but contributed significantly to restoring 15‐LOX‐1 expression and inducing apoptosis by NS‐398 or sodium butyrate. Maintaining 15‐LOX‐1 transcriptional silencing in cancer cells is a multifactorial process involving GATA‐6 overexpression and other regulatory events.Shureiqi, I., Zuo, X., Broaddus, R., Wu, Y., Guan, B., Morris, J. S., Lippman, S. M. The transcription factor GATA‐6 is overexpressed in vivo and contributes to silencing 15‐LOX‐1 in vitro in human colon cancer FASEB J. 21, 743–753 (2007)

Targeted Genetic Disruption of Peroxisome Proliferator–Activated Receptor-δ and Colonic Tumorigenesis

Peroxisome proliferator-activated receptor-delta (PPAR-delta) is overexpressed in human colon cancer, but its contribution to colonic tumorigenesis is controversial. We generated a mouse model in which PPAR-delta was genetically disrupted in colonic epithelial cells by targeted deletion of exon 4. Elimination of colon-specific PPAR-delta expression was confirmed by real-time reverse transcription-polymerase chain reaction (real-time RT-PCR), immunoblotting, and activity assays. Mice with and without targeted PPAR-delta genetic disruption (10-11 mice per group) were tested for incidence of azoxymethane-induced colon tumors. The effects of targeted PPAR-delta deletion on vascular endothelial growth factor expression were determined by real-time RT-PCR. Targeted PPAR-delta genetic disruption inhibited colonic carcinogenesis: Mice with PPAR-delta((-/-)) colons developed 98.5% fewer tumors than wild-type mice (PPAR-delta((-/-)) vs wild-type, mean = 0.1 tumors per mouse vs 6.6 tumors per mouse, difference = 6.5 tumors per mouse, 95% confidence interval = 4.9 to 8.0 tumors per mouse, P < .001, two-sided test). Increased expression of vascular endothelial growth factor in colon tumors vs normal colon was suppressed by loss of PPAR-delta expression. These findings indicate that PPAR-delta has a crucial role in promoting colonic tumorigenesis.

Profiling Lipoxygenase Metabolism in Specific Steps of Colorectal Tumorigenesis

Lipoxygenases (LOX) are key enzymes for the oxidative metabolism of polyunsaturated fatty acids into biologically active products. Clinical data on comparative levels of various LOX products in tumorigenesis are lacking. Therefore, we examined the profiles of several LOX products (5-LOX, 12-LOX, 15-LOX-1, and 15-LOX-2) by liquid chromatography/tandem mass spectrometry in the major steps of colorectal tumorigenesis (normal, polyp, and cancer) in a clinical study of 125 subjects (49 with normal colon, 36 with colorectal polyps, and 40 with colorectal cancer) who underwent prospective colorectal biopsies to control for various potential confounding factors (e.g., diet, medications). Mean 13-hydroxyoctadecadienoic acid (13-HODE) levels were significantly higher in normal colon [mean, 36.11 ng/mg protein; 95% confidence interval (95% CI), 31.56-40.67] than in paired colorectal cancer mucosa (mean, 27.01 ng/mg protein; 95% CI, 22.00-32.02; P = 0.0002), and in normal colon (mean, 37.15 ng/mg protein; 95% CI, 31.95-42.34) than in paired colorectal polyp mucosa (mean, 28.07 ng/mg protein; 95% CI, 23.66-32.48; P < 0.001). Mean 13-HODE levels, however, were similar between the left (mean, 37.15 ng/mg protein; 95% CI, 31.95-42.35) and the right normal colon (mean, 32.46 ng/mg protein; 95% CI, 27.95-36.98; P = 0.09). No significant differences with regard to 12- or 15-hydroxyeicosatetraenoic acid or leukotriene B4 levels were detected between normal, polyp, and cancer mucosae. 15-LOX-1 inhibited interleukin-1β expression. This study establishes that reduced 13-HODE levels are a specific alteration in the LOX product profile associated with human colorectal tumorigenesis. Cancer Prev Res; 3(7); 829–38. ©2010 AACR.

KLF4 Is Essential for Induction of Cellular Identity Change and Acinar-to-Ductal Reprogramming during Early Pancreatic Carcinogenesis

Understanding the molecular mechanisms of tumor initiation has significant impact on early cancer detection and intervention. To define the role of KLF4 in pancreatic ductal adenocarcinoma (PDA) initiation, we used molecular biological analyses and mouse models of klf4 gain- and loss-of-function and mutant Kras. KLF4 is upregulated in and required for acinar-to-ductal metaplasia. Klf4 ablation drastically attenuates the formation of pancreatic intraepithelial neoplasia induced by mutant Kras(G12D), whereas upregulation of KLF4 does the opposite. Mutant KRAS and cellular injuries induce KLF4 expression, and ectopic expression of KLF4 in acinar cells reduces acinar lineage- and induces ductal lineage-related marker expression. These results demonstrate that KLF4 induces ductal identity in PanIN initiation and may be a potential target for prevention of PDA initiation.

15-Lipoxygenase-1 as a tumor suppressor gene in colon cancer: is the verdict in?

Abstract15-Lipoxygenase-1 (15-LOX-1) is an inducible and highly regulated enzyme in normal human cells that plays a key role in the production of lipid signaling mediators, such as 13-hydroxyoctadecadienoic acid (13-HODE) from linoleic acid. 15-LOX-1 significantly contributes to the resolution of inflammation and to the terminal differentiation of normal cells. 15-LOX-1 is downregulated in human colorectal polyps and cancers. Emerging data support a tumor suppressor role for 15-LOX-1, especially in colon cancer. These data indicate that 15-LOX-1 promotes various anti-tumorigenic events, including cell differentiation and apoptosis, and inhibits chronic inflammation, angiogenesis, and metastasis. The transcriptional repression of 15-LOX-1 in colon cancer cells is complex and involves multiple mechanisms (e.g., histone methylation, transcriptional repressor binding). Re-expression of 15-LOX-1 in colon cancer cells can function as an important therapeutic mechanism and could be further exploited to develop novel treatment approaches for this common cancer.

Mechanistic Contribution of Ubiquitous 15-Lipoxygenase-1 Expression Loss in Cancer Cells to Terminal Cell Differentiation Evasion

Loss of terminal cell differentiation promotes tumorigenesis. 15-Lipoxygenase-1 (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 nontransformed bronchial epithelial but not in lung cancer cell lines. 15-LOX-1 expression levels were lower in human tumors than in 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 reexpression to inhibit tumorigenesis. Cancer Prev Res; 4(12); 1961–72. ©2011 AACR.

Effects of Gut-Targeted 15-LOX-1 Transgene Expression on Colonic Tumorigenesis in Mice

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.