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Background Pancreatic ductal adenocarcinoma (PDA) has a poor prognosis largely due to its therapeutic resistance. Inactivation of vitamin D/vitamin D receptor (VDR) signaling contributes to the malignant phenotype of PDA, and altered expression of oncoprotein mucin 1 (MUC1) is involved in drug resistance of cancer cells. In the present study, we determined whether vitamin D/VDR signaling regulates the expression and function of MUC1 and its effect on gemcitabine resistance of pancreatic cancer cells. Methods We performed an unbiased reverse-phase protein array (RPPA) to identify vitamin D3 downstream molecules in PDA cells. We used molecular analyses and animal models to determine the impact of vitamin D/VDR signaling on MUC1 expression and response to gemcitabine treatment. Results RPPA analysis indicated that MUC1 protein expression was significantly reduced in human PDA cells after treatment with vitamin D3 or its analog calcipotriol. VDR negatively regulated MUC1 expression in both gain- and loss-of-function assays. Vitamin D3 or its analog significantly induced VDR and inhibited MUC1 expression in AsPC1-GemR and Colo357-GemR gemcitabine-resistant cells and sensitized the resistant cells to gemcitabine treatment. siRNA knockdown experiments indicated that inhibition of MUC1 expression was essential for calcipotriol-associated sensitization of PDA cells to gemcitabine treatment in vitro. Administration of the vitamin D3 analog paricalcitol significantly enhanced the therapeutic efficacy of gemcitabine in xenograft and orthotopic mouse models and increased the intratumoral concentration of dFdCTP, the active gemcitabine metabolite. Conclusions These findings demonstrate a previously unidentified vitamin D/VDR-MUC1 signaling axis involved in the regulation of gemcitabine resistance in PDA, and suggests that combinational therapies that include targeted activation of vitamin D/VDR signaling may improve the outcomes of patients with PDA.

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.

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Supplementary Figure 1 from Profiling Lipoxygenase Metabolism in Specific Steps of Colorectal Tumorigenesis

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Why celecoxib exerts chemopreventive activity in only some familial adenomatous polyposis (FAP) patients remains poorly understood. We conducted a phase II clinical study to identify potential predictive biomarkers for celecoxib chemopreventive activity in FAP. Twenty-seven patients with FAP completed a 6-month oral course of 400 mg of celecoxib twice a day; they underwent colonoscopies before and after celecoxib treatment to assess colorectal polyp tumor burden and to obtain normal and polyp colorectal biopsies to measure celecoxib, 13-S-hydroxyoctadecadienoic acid (13-HODE), 15-HETE, 12-HETE, and LTB4 levels by LC/MS-MS. Celecoxib levels in sera from those patients were also measured before treatment and after 2, 4, and 6 months of treatment. Nineteen of the 27 patients experienced a response to celecoxib, with a ≥ 28% reduction of colonic polyp burden on the basis of a reproducible quantitative assessment of colonoscopy results. Celecoxib levels were significantly lower in polyp tissues than in normal colorectal tissues. Celecoxib levels in sera and normal colorectal tissues were correlated in patients who experienced a response to celecoxib but not in those who did not. Among the measured lipoxygenase products, only 13-HODE levels were significantly lower in polyp tissues than in normal tissues. Our findings demonstrate the differential bioavailability of celecoxib between normal and polyp tissues and its potential effects on clinical response in patients with FAP.Prevention Relevance:This study evaluated potential predictive biomarkers for celecoxib chemopreventive activity in patients with FAP. Our findings demonstrated the differential bioavailability of celecoxib between normal and polyp tissues and its potential effects on clinical chemopreventive response in patients with FAP.See related Spotlight, p. 205

Supplementary Data from Celecoxib Colorectal Bioavailability and Chemopreventive Response in Patients with Familial Adenomatous Polyposis

One file in MS word format contains the following information on methods: 1. Flow Cytometric Cell Sorting Analysis; 2. Reverse Transcription and Quantitative Real-Time PCR; 3. Western Blot Analysis; 4. ChIP Assay; 5. Immunocytochemistry; 6. Promoter Activity Analysis; 7. Transient transfection of Plasmids and siRNAs; 8. Cell Scratch-Wound Healing Assay; 9. Cell Invasion Assay; 10. Immunohistochemistry Analysis; 11. Animal Models of Tumorigenesis and Metastasis; 12. SUPPLEMENTAL REFERENCES

KLF4 and CD44 regulate cancer cell stemness, but their precise functions and roles in metastatic progression are not well understood. In this study, we used both inducible and genetic engineering approaches to assess whether the activities of these two factors intersect in pancreatic cancer. We found that genetic ablation of Klf4 in pancreatic cancer cells isolated from Klf4flox/flox mice drastically increased CD44 expression and promoted the acquisition of stem-like properties, whereas tetracycline-inducible expression of KLF4 suppressed these properties in vitro and in vivo. Further mechanistic investigation revealed that KLF4 bound to the CD44 promoter to negatively regulate transcription and also the expression of the CD44 variant. Moreover, in human pancreatic ductal adenocarcinoma (PDAC) tissues, the expression patterns of KLF4 and CD44 were mutually exclusive, and this inverse relationship was particularly striking in human metastatic pancreatic tumors and in autochthonous mouse models of PDAC. Taken together, our findings demonstrate that KLF4 acts as a tumor suppressor in PDAC cells that restricts metastatic behaviors through direct negative regulation of CD44, providing support for the clinical investigation of therapeutic approaches focusing on targeted KLF4 activation in advanced tumors. Cancer Res; 76(8); 2419–31. ©2016 AACR.

Purpose: The dismal prognosis of pancreatic cancer has been linked to poor tumor differentiation. However, molecular basis of pancreatic cancer differentiation and potential therapeutic value of the underlying molecules remain unknown. We investigated the mechanistic underexpression of Krüppel-like factor 4 (KLF4) in pancreatic cancer and defined a novel epigenetic pathway of its activation for pancreatic cancer differentiation and treatment.Experimental Design: Expressions of KLF4 and DNMT1 in pancreatic cancer tissues were determined by IHC and the genetic and epigenetic alterations of KLF4 in and KLF4′s impact on differentiation of pancreatic cancer were examined using molecular biology techniques. The function of dietary 3,3′-diindolylmethane (DIM) on miR-152/DNMT1/KLF4 signaling in pancreatic cancer was evaluated using both cell culture and animal models.Results: Overexpression of DNMT1 and promoter hypermethylation contributed to decreased KLF4 expression in and associated with poor differentiation of pancreatic cancer. Manipulation of KLF4 expression significantly affected differentiation marker expressions in pancreatic cancer cells. DIM treatment significantly induced miR-152 expression, which blocked DNMT1 protein expression and its binding to KLF4 promoter region, and consequently reduced promoter DNA methylation and activated KLF4 expression in pancreatic cancer cells. In addition, DIM treatment caused significant inhibition of cell growth in vitro and tumorigenesis in animal models of pancreatic cancer.Conclusions: This is the first demonstration that dysregulated KLF4 expression associates with poor differentiation of pancreatic cancer. Epigenetic activation of miR-152/DNMT1/KLF4 signaling pathway by dietary DIM causes differentiation and significant growth inhibition of pancreatic cancer cells, highlighting its translational implications for pancreatic and other cancers. Clin Cancer Res; 23(18); 5585–97. ©2017 AACR.

Supplementary Figure 1. Genetic and epigenetical alterations of KLF4 in pancreatic cancer; Supplementary Figure 2. A demonstration of LCM/ DNA methylation specific PCR analysis of KLF4 promoter methylation; Supplementary Figure 3. Comparison of DNMT1 and KLF4 expressions in pancreatic tissues determined by IHC analysis; Supplementary Figure 4. The effect of DIM treatment on cell cycle progression; Supplementary Figure 5. Spheroid colony formation and tumorigenesis; Supplementary Figure 6. Tumorigenesis assay using orthotopic pancreatic cancer mouse model; Supplementary Figure 7. The effect of DIM treatment on gene expression in pancreatic cancer cells; Supplementary Figure 8. The effects of DIM and miR-152 on gene expression.

Supplementary Methods

APC mutations activate aberrant β-catenin signaling to drive initiation of colorectal cancer; however, colorectal cancer progression requires additional molecular mechanisms. PPAR-delta (PPARD), a downstream target of β-catenin, is upregulated in colorectal cancer. However, promotion of intestinal tumorigenesis following deletion of PPARD in Apcmin mice has raised questions about the effects of PPARD on aberrant β-catenin activation and colorectal cancer. In this study, we used mouse models of PPARD overexpression or deletion combined with APC mutation (ApcΔ580) in intestinal epithelial cells (IEC) to elucidate the contributions of PPARD in colorectal cancer. Overexpression or deletion of PPARD in IEC augmented or suppressed β-catenin activation via up- or downregulation of BMP7/TAK1 signaling and strongly promoted or suppressed colorectal cancer, respectively. Depletion of PPARD in human colorectal cancer organoid cells inhibited BMP7/β-catenin signaling and suppressed organoid self-renewal. Treatment with PPARD agonist GW501516 enhanced colorectal cancer tumorigenesis in ApcΔ580 mice, whereas treatment with PPARD antagonist GSK3787 suppressed tumorigenesis. PPARD expression was significantly higher in human colorectal cancer–invasive fronts versus their paired tumor centers and adenomas. Reverse-phase protein microarray and validation studies identified PPARD-mediated upregulation of other proinvasive pathways: connexin 43, PDGFRβ, AKT1, EIF4G1, and CDK1. Our data demonstrate that PPARD strongly potentiates multiple tumorigenic pathways to promote colorectal cancer progression and invasiveness.Significance:These findings address long-standing, important, and unresolved questions related to the potential role of PPARD in APC mutation-dependent colorectal tumorigenesis by showing PPARD activation enhances APC mutation-dependent tumorigenesis.

Supplementary Table 1. Patients' Characteristics and KLF4 Expression; Supplementary Table 2. Patients' Characteristics and DNMT1 Expression; Table 3. Upregulated microRNAs after DIM Treatment; Table 4. Down-regulated microRNAs after DIM Treatment.

Supplementary Figure 1-7 Figure S1. Effects of KLF4 overexpression on tumorsphere formation and liver metastasis of cancer stem-like cells; Figure S2. Effect of Klf4 gene disruption on cell morphology of mouse pancreatic cancer cells; Figure S3. IHC staining of Klf4 and CD44 expressions in intestinal tissues Figure S4. KLF4 and CD44 expressions in PANC-1 cells; Figure S5. KLF4 inhibits CD44v and ESRP-1 expressions in pancreatic and colon cancer cells; Figure S6. KLF4 expression correlates with metastatic potential in human cancer cells; Figure S7. KLF4/CD44 expressions in primary and metastatic tumor tissues

Supplemental Figure Legends Figure S1. Effects of KLF4 overexpression on tumorsphere formation and liver metastasis of cancer stem-like cells; Figure S2. Effect of Klf4 gene disruption on cell morphology of mouse pancreatic cancer cells; Figure S3. IHC staining of Klf4 and CD44 expressions in intestinal tissues; Figure S4. KLF4 and CD44 expressions in PANC-1 cells; Figure S5. KLF4 inhibits CD44v and ESRP-1 expressions in pancreatic and colon cancer cells; Figure S6. KLF4 expression correlates with metastatic potential in human cancer cells; Figure S7. KLF4/CD44 expressions in primary and metastatic tumor tissues

Supplementary Figures S1-S7 - This file includes supplementary figures S1-S7: Figure S1. PPARD increased active β-catenin and its target gene (e.g. c-Myc and cyclin D1) expression levels in mouse IECs and human HCT116 colorectal cancer cells by western blot and qRT-PCR. Figure S2. PPARD and its ligand GW501516 promoted intestinal tumorigenesis in multiple APC mutant mouse models, shown by intestinal photographs and tumor number counts. Figure S3. Western blot and IHCs showing that PPARD increased BMP7/TAK1/active β-catenin expression levels in mouse IECs. Figure S4. IHC microphotographs and score results showing active β-catenin expression levels in 41 human paired colorectal adenomas (Adenoma), CRC tumor centers (Tumor center), and cancer invasive fronts (Invasive front). Figure S5. Western blot and IHC results showing that PPARD upregulated AKT1 but not AKT2 to increase p-rpS6 levels in mouse IECs and human HCT116 colorectal cancer cells. Figure S6. Immunofluorescence staining of rRNA, IHC microphotographs and IHC score results of CDK1 showing rRNA expression levels in mouse intestinal tissues from PD and ApcÎ"580-PD mice and their corresponding control littermates, and nuclear CDK1 expression levels in 41 human paired colorectal adenomas (Adenoma), CRC tumor centers (Tumor center), and cancer invasive fronts (Invasive front). Figure S7. Multiple human public database analyses showing PPARD and BMP7 genetic alterations in colon cancer patients, and comparison of the survival probability for the colon cancer patients with low and high expression of BMP7.

Supplementary Tables Table S1. CD44 expression and clinicopathologic characteristics; Table S2. KLF4 expression and clinicopathologic characteristics; Table S3. Specific primers for target and control genes; Table S4. Antibodies for target and control protein expression in Western blot analyses

APC mutations activate aberrant β-catenin signaling to drive initiation of colorectal cancer; however, colorectal cancer progression requires additional molecular mechanisms. PPAR-delta (PPARD), a downstream target of β-catenin, is upregulated in colorectal cancer. However, promotion of intestinal tumorigenesis following deletion of PPARD in Apcmin mice has raised questions about the effects of PPARD on aberrant β-catenin activation and colorectal cancer. In this study, we used mouse models of PPARD overexpression or deletion combined with APC mutation (ApcΔ580) in intestinal epithelial cells (IEC) to elucidate the contributions of PPARD in colorectal cancer. Overexpression or deletion of PPARD in IEC augmented or suppressed β-catenin activation via up- or downregulation of BMP7/TAK1 signaling and strongly promoted or suppressed colorectal cancer, respectively. Depletion of PPARD in human colorectal cancer organoid cells inhibited BMP7/β-catenin signaling and suppressed organoid self-renewal. Treatment with PPARD agonist GW501516 enhanced colorectal cancer tumorigenesis in ApcΔ580 mice, whereas treatment with PPARD antagonist GSK3787 suppressed tumorigenesis. PPARD expression was significantly higher in human colorectal cancer–invasive fronts versus their paired tumor centers and adenomas. Reverse-phase protein microarray and validation studies identified PPARD-mediated upregulation of other proinvasive pathways: connexin 43, PDGFRβ, AKT1, EIF4G1, and CDK1. Our data demonstrate that PPARD strongly potentiates multiple tumorigenic pathways to promote colorectal cancer progression and invasiveness. Significance: These findings address long-standing, important, and unresolved questions related to the potential role of PPARD in APC mutation-dependent colorectal tumorigenesis by showing PPARD activation enhances APC mutation-dependent tumorigenesis.

Objective: Pancreatic ductal adenocarcinoma (PDA) has a dismal prognosis, largely due to its resistance to currently available therapies. Identifying the determinants of and signaling pathways regulating this resistance is essential for the development of effective therapeutic modalities to improve the outcome of PDA. The purpose of this study is to define the role of vitamin D/VDR signaling and underlying mechanisms in PDA pathogenesis and therapeutic resistance. Materials & Methods: We used an unbiased reverse phase protein array (RPPA) to identify Vitamin D3 downstream molecules; we also used a variety of cellular and molecular biological approaches to determine the impact of vitamin D/VDR signaling on the expression of MUC1 mucin and the cellular response to gemcitabine treatment in PDA cells. We used in vivo experimental systems to determine the effect of combination use of gemcitabine with Vitamin D analogue on pancreatic tumorigenicity. Additionally, we used ultra-high resolution mass spectrometry (HRMS) to analyze the relative abundance of intracellular dFdCTP in tumor tissue samples. Results: We identified that MUC1 protein expression was significantly reduced in AsPC-1 human pancreatic cancer cells after treatment with vitamin D3 or its analogue calcipotriol (Cal) by the RPPA analysis, which was further confirmed in MiaPaca-2 and Colo357 pancreatic cancer cells. VDR negatively regulated MUC1 expression in pancreatic cancer cells in both gain- and loss-of-function assays. We established gemcitabine (Gem) resistant AsPC-1-GemR and Colo357-GemR cells and found that AsPC-1-GemR and Colo357-GemR cells had much lower levels of VDR but higher levels of MUC1 protein expression compared to their parental cells, Vitamin D3 or its analogue significantly induced VDR and inhibited MUC1 expression in AsPC-1-GemR and Colo357-GemR cells, and sensitized the resistant cells to gemcitabine treatment. The results of siRNA knockdown experiments indicated that inhibition of MUC1 expression was essential for Cal’s sensitization of PDA cells to Gem treatment in vitro. In keeping with these findings, administration of the Vitamin D3 analogue, paricalcitol (PA) significantly enhanced the therapeutic efficacy of Gem compared to chemotherapy alone in xenograft and syngeneic mouse models of pancreatic cancer without any significant systemic toxicity. The improved combination treatment correlated with increased intratumoral concentration of dFdCTP, the biological active metabolite of gemcitabine. Conclusions: This study reveals a previously unidentified vitamin D/VDR-MUC1 signaling pathway involved in the regulation of gemcitabine resistance of PDA. Our results suggest that combinational therapies which include targeted activation of vitamin D/VDR signaling may improve the outcomes of patients with PDA. Citation Format: Daoyan Wei, Margarete Hafley, Liang Wang, Yi Liu, Peiying Yang, Xiangsheng Zuo, Robert S. Bresalier. Activation of vitamin D/VDR signaling reverses gemcitabine resistance of pancreatic cancer cells through inhibition of MUC1 expression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6367.

Ample evidence from epidemiological studies links high-sugar diets with increased breast cancer risk, but the underlying molecular mechanisms remain unclear. We previously reported that sucrose-enriched diets (SED) accelerated breast tumorigenesis in MMTV-ErbB2 mice and promoted the breast tumor growth in orthotopic models of mouse (4T1) or human breast cancer cells (MDA-MB-231) by up-regulating 12-lipoxygenase (12-LOX) and its arachidonate metabolite 12-HETE. In this report, we performed mechanistic studies to determine the role of 12-LOX in SED-induced breast tumorigenesis and also examined the SED effects on remodeling breast tumor microenvironment during SED-induced breast tumorigenesis. We found SED (diet with 125 g/kg sucrose), at a concentration equivalent to the average sugar consumption of American population, significantly increased the 4T1 orthotopic tumor weights by 3.7-fold in average compared to the isocaloric cornstarch control diet group. We obtained data showing that 12-LOX downregulation by CRISPR/Cas9 technology not only significantly reduced tumor incidence and inhibited the tumor growth, but also blocked the SED-promoted breast tumor growth in the MDA-MB-231 cell mouse orthotopic model. We also found that SED increased expression of peroxisome proliferator-activated receptor-delta (PPARd), a lipid nuclear receptor along with its target genes (i. g., LPL, CD36 and SCD1) in 4T1 orthotopic breast tumor tissues. 12-LOX downregulation decreased PPARD expression in MDA-MB-231 cells, while 12-HETE increased PPARd and its target proteins such as PDK4 and ANGPLT4 expression levels in 4T1 cells. Together, these data suggest PPARd as a downstream target gene of 12-LOX. Additionally, our studies showed that SED significantly increased chemokine CCL2 in 4T1 orthotopic tumor tissues and 12-HETE led to higher secreted CCL2 in 4T1 mouse breast cancer cells which was blocked by suppressing PPARd expression. Furthermore, we observed 10-fold higher CD11b+/Ly6G+ cells in SED-induced 4T1 tumor tissues by flow cytometry. Among CD45+ cells, percentage of CD8+ T cells was decreased by 34% in SED-induced 4T1 tumors, and percentages of CD8+/CD69+ (active CD8+ T cells) and CD8+/GrzB+ (effector T cells) were reduced by 78% and 60%, respectively, in SED-induced 4T1 tumors, suggesting SED also led to immune suppressive tumor microenvironment (iTME) during breast tumorigenesis. We further noted that MDSCs, but not macrophages, expressed higher CCR2 in TILs of SED fed tumor than that of cornstarch control diet group, indicating SED induced accumulation of MDSCs is likely mediated by CCL2. In conclusion, our data strongly supports added sugar (sucrose) to accelerate the development and progression of breast cancer, potentially involving up-regulation of the expression of 12-LOX and PPARd as well as iTME. Citation Format: Sharmistha Chakraborty, Xiangsheng Zuo, Venkatesh Hedge, Megan Tran, Yan Jiang, Mihai Gagea, Jagannadha Sastry, Peiying Yang. Dietary sugar promotes breast tumorigenesis partially through upregulating 12 lipoxygenase/PPARd signaling and remodeling breast tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5954.

Pancreatic intraepithelial neoplasia (PanIN) is a precursor of pancreatic ductal adenocarcinoma (PDAC), which commonly occurs in the general populations with aging. Although most PanIN lesions (PanINs) harbor oncogenic KRAS mutations that initiate pancreatic tumorigenesis, PanINs rarely progress to PDAC. Critical factors that promote this progression, especially targetable ones, remain poorly defined. We show that peroxisome proliferator-activated receptor-delta (PPARD), a lipid nuclear receptor, is upregulated in PanINs in humans and mice. Furthermore, PPARD ligand activation by a high-fat diet or GW501516 (a highly selective, synthetic PPARD ligand) in mutant KRASG12D (KRASmu) pancreatic epithelial cells strongly accelerates PanIN progression to PDAC. This PPARD activation induces KRASmu pancreatic epithelial cells to secrete CCL2, which recruits immunosuppressive macrophages and myeloid-derived suppressor cells into pancreas via the CCL2/CCR2 axis to orchestrate an immunosuppressive tumor microenvironment and subsequently drive PanIN progression to PDAC. Our data identify PPARD signaling as a potential molecular target to prevent PDAC development in subjects harboring PanINs. Citation Format: Yi Liu, Yasunori Deguchi, Daoyan Wei, Micheline J. Moussalli, Fuyao Liu, Eriko Deguchi, Donghui Li, Huamin Wang, Lovie Ann Valentin, Jennifer K. Colby, Jing Wang, Xiaofeng Zheng, Haoqiang Ying, Mihai Gagea, Baoan Ji, Jiaqi Shi, James C. Yao, Xiangsheng Zuo, Imad Shureiqi. Rapid acceleration of KRAS-mutant pancreatic carcinogenesis via remodeling of tumor immune microenvironment by PPARD [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3821.

Pro-obesity high-fat diet is linked with an increased incidence of pancreatic cancers, but the molecular underpinnings of this association remain poorly understood. Here, we report that PPARD is upregulated in pancreatic intraepithelial neoplasia lesions (PanINs) at early stages of pancreatic tumorigenesis in humans and mutant Kras mice. Transgenic overexpression of Ppard in pancreatic epithelial cells drastically accelerates the development and progression of pancreatic ductal adenocarcinoma in mutant Kras mice when activated by feeding the mice with a high-fat diet or a diet containing GW501516 (50 mg/kg), a selective PPARD agonist. In contrast, pancreatic Ppard genetic deletion significantly suppressed the promotion of pancreatic tumorigenesis by these diets. Mechanistically, we found that this Ppard hyperactivation in pancreatic epithelial cells of mutant Kras mice increased production of chemokines and cytokines (e.g., CcI2, CcI4-5, CxcI5 and II6), leading to the robustly increased recruitment of myeloid-derived suppressor cells and macrophages into pancreata, which fostered an immune suppressive microenvironment and subsequently accelerated pancreatic ductal adenocarcinoma development and progression. Our findings demonstrate that PPARD plays an essential role in the promotion of pancreatic tumorigenesis by a high-fat diet. Targeted inhibition of PPARD activation is a potential interventive strategy for pancreatic cancer prevention and therapy.

Why celecoxib exerts chemopreventive activity in only some familial adenomatous polyposis (FAP) patients remains poorly understood. We conducted a phase II clinical study to identify potential predictive biomarkers for celecoxib chemopreventive activity in FAP. Twenty-seven patients with FAP completed a 6-month oral course of 400 mg of celecoxib twice a day; they underwent colonoscopies before and after celecoxib treatment to assess colorectal polyp tumor burden and to obtain normal and polyp colorectal biopsies to measure celecoxib, 13-S-hydroxyoctadecadienoic acid (13-HODE), 15-HETE, 12-HETE, and LTB4 levels by LC/MS-MS. Celecoxib levels in sera from those patients were also measured before treatment and after 2, 4, and 6 months of treatment. Nineteen of the 27 patients experienced a response to celecoxib, with a ≥ 28% reduction of colonic polyp burden on the basis of a reproducible quantitative assessment of colonoscopy results. Celecoxib levels were significantly lower in polyp tissues than in normal colorectal tissues. Celecoxib levels in sera and normal colorectal tissues were correlated in patients who experienced a response to celecoxib but not in those who did not. Among the measured lipoxygenase products, only 13-HODE levels were significantly lower in polyp tissues than in normal tissues. Our findings demonstrate the differential bioavailability of celecoxib between normal and polyp tissues and its potential effects on clinical response in patients with FAP.Prevention Relevance:This study evaluated potential predictive biomarkers for celecoxib chemopreventive activity in patients with FAP. Our findings demonstrated the differential bioavailability of celecoxib between normal and polyp tissues and its potential effects on clinical chemopreventive response in patients with FAP.See related Spotlight, p. 205

Simple Summary Pancreatic cancer is an almost universally lethal cancer, largely due to its late diagnosis, early metastasis, and therapeutic resistance. This highlights the need to develop novel and effective intervention strategies to improve the outcomes of patients with pancreatic cancer. Vitamin D is one of the hottest topics in cancer research and clinics because of its pleiotropic functions on the hallmarks of cancer. Here we critically review past and current efforts that define the effects of vitamin D on the risk, incidence, patient survival, and mortality of pancreatic cancer. We also provide overviews on the opportunities and challenges associated with vitamin D as an economic adjunct to improve the efficacy of immunotherapy and chemo- or radiotherapy for pancreatic cancer. Abstract Pancreatic cancer has a dismal prognosis, while its incidence is increasing. This is attributed, in part, to a profound desmoplastic and immunosuppressive tumor microenvironment associated with this cancer and resistance to current available therapies. Novel and effective intervention strategies are urgently needed to improve the outcomes of patients with pancreatic cancer. Vitamin D has pleiotropic functions beyond calcium–phosphate homeostasis and has been extensively studied both in the laboratory and clinic as a potential preventive agent or adjunct to standard therapies. Accumulating evidence from ecological, observational, and randomized controlled trials suggests that vitamin D has beneficial effects on risk, survival, and mortality in pancreatic cancer, although controversies still exist. Recent advances in demonstrating the important functions of vitamin D/vitamin D receptor (VDR) signaling in the regulation of stromal reprogramming, the microbiome, and immune response and the emergence of checkpoint immunotherapy provide opportunities for using vitamin D or its analogues as an adjunct for pancreatic cancer intervention. Many challenges lie ahead before the benefits of vitamin D can be fully realized in pancreatic cancer. These challenges include the need for randomized controlled trials of vitamin D to assess its impact on the risk and survival of pancreatic cancer, optimizing the timing and dosage of vitamin D or its analogues as an adjunct for pancreatic cancer intervention and elucidating the specific role of vitamin D/VDR signaling in the different stages of pancreatic cancer. Nevertheless, vitamin D holds great promise for reducing risk and improving outcomes of this disease.

Mounting evidence supports a mechanistic link between inflammation and cancer, especially colon cancer. ALOX15 (15-lipoxygenase-1) plays an important role in the formation of key lipid mediators (e.g., lipoxins and resolvins) to terminate inflammation. ALOX15 expression is downregulated in colorectal cancer (CRC). Intestinally-targeted transgenic expression of ALOX15 in mice inhibited dextran sodium sulfate-induced colitis from promoting azoxymethane- induced colorectal tumorigenesis, demonstrating that ALOX15 can suppress inflammation-driven promotion of carcinogen-induced colorectal tumorigenesis and therefore ALOX15 downregulation during tumorigenesis is likely to enhance the link between colitis and colorectal tumorigenesis. ALOX15 suppressed the TNF-α, IL-1β/NF-κB, and IL-6/STAT3 signaling pathways, which play major roles in promotion of colorectal cancer by chronic inflammation. Defining ALOX15’s regulatory role in colitis-associated colorectal cancer could identify important molecular regulatory events that could be targeted to suppress promotion of tumorigenesis by chronic inflammation.

SUMMARYAberrant Wnt/β-catenin activation is a major driver of colorectal cancer (CRC), which is typically initiated byAPCmutations. Additional modifiable factors beyondAPCmutations have been recognized to be important for further potentiation of aberrant β-catenin activation to promote colorectal tumorigenesis. These factors have yet to be clearly identified. Western-type diets are increasingly enriched in linoleic acid (LA). LA-enriched diet however promotes chemically-induced colorectal tumorigenesis in rodent models. Furthermore, the main metabolizing enzyme of LA, 15-lipoxygenase-1 (15-LOX-1), is transcriptionally silenced in CRC. Whether LA and 15-LOX-1 affect Wnt/β-catenin signaling to modulate colorectal tumorigenesis is poorly understood. Herein, we report that high dietary LA promoted colorectal tumorigenesis in mice with intestinally targetedAPCmutation (ApcΔ580) by upregulating a Wnt receptor, LRP5 expression, and β-catenin activation. 15-LOX-1 transgenic expression in intestinal epithelial cells suppressed LRP5 expression, β-catenin activation and subsequently CRC in these mice. In particular, 15-LOX-1 peroxidation of LA in phosphatidylinositol-3-phosphates (PI3P_LA) into PI3P_13-HODE decreased PI3P binding to SNX17and LRP5, which inhibited LRP5 recycling from endosomes to the plasma membrane, thereby leading to an increase of LRP5 lysosomal degradation. Our findings demonstrate for the first time that 15-LOX-1 metabolism of LA in PI3P to regulate LRP5 membrane abundance is a modifiable factor of Wnt/β-catenin aberrant signaling that could be potentially therapeutically targeted to suppress colorectal tumorigenesis and progression.

APC mutation activation of Wnt/β-catenin drives initiation of colorectal carcinogenesis (CRC). Additional factors potentiate β-catenin activation to promote CRC. Western diets are enriched in linoleic acid (LA); LA-enriched diets promote chemically induced CRC in rodents. 15-Lipoxygenase-1 (15-LOX-1), the main LA-metabolizing enzyme, is transcriptionally silenced during CRC. Whether LA and 15-LOX-1 affect Wnt/β-catenin signaling is unclear. We report that high dietary LA promotes CRC in mice treated with azoxymethane or with an intestinally targeted Apc mutation (Apc(Δ580)) by upregulating Wnt receptor LRP5 protein expression and β-catenin activation. 15-LOX-1 transgenic expression in mouse intestinal epithelial cells suppresses LRP5 protein expression, β-catenin activation, and CRC. 15-LOX-1 peroxidation of LA in phosphatidylinositol-3-phosphates (PI3P_LA) leads to PI3P_13-HODE formation, which decreases PI3P binding to SNX17 and LRP5 and inhibits LRP5 recycling from endosomes to the plasma membrane, thereby increasing LRP5 lysosomal degradation. This regulatory mechanism of LRP5/Wnt/β-catenin signaling could be therapeutically targeted to suppress CRC.

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.

Western-type diets have been increasingly enriched with linoleic acid (LA). The impact of excess dietary LA on cancer risk remains poorly understood although some studies suggest that high LA diets promote chemically-induced colorectal carcinogenesis (CRC) in rodent models. 15-lipoxygenase-1 (15-LOX-1), the main metabolizing enzyme of LA, is transcriptionally silenced during the early stages of CRC. Whether 15-LOX-1 impacts the effects of excess LA on CRC is unknown. Herein, we report that high dietary LA promoted CRC in intestinally targeted APC mutation (ApcΔ580) and azoxymethane-induced CRC mouse models by upregulating expression of LRP5, a Wnt receptor, and aberrant β-catenin activation. These effects were negatively regulated by 15-LOX-1 when this enzyme was transgenically expressed in intestinal epithelial cells. Complementary genetic deletion studies of 15-LOX-1 in mice further supported 15-LOX-1's suppressive effects on these events. Mechanistic studies showed that 15-LOX-1 peroxidation of LA in phosphatidylinositol-3-phosphates (PI3P_LA) into PI3P_13-HODE decreased PI3P binding to SNX17 and LRP5, which inhibited LRP5 recycling from endosomes to the plasma membrane, thereby leading to an increase of LRP5 lysosomal degradation. Our findings demonstrate for the first time the importance of linoleic acid metabolism by 15-LOX-1 within complex lipids such as PI3P in regulating LRP5 membranous abundance and subsequently Wnt/β-catenin aberrant signaling activation and CRC risk. These results strongly support the need to carefully consider the potential of increasing CRC risk by enriching the diets with LA especially for individuals with low 15-LOX-1 expression. Citation Format: Fuyao Liu, Xiangsheng Zuo, Yi Liu, Yasunori Deguchi, Micheline J. Moussalli, Weidong Chen, Peiying Yang, Bo Wei, Lin Tan, Philip L. Lorenzi, Shen Gao, Jonathan C. Jaoude, Amir Mehdizadeh, Lovie A. Valentin, Daoyan Wei, Imad Shureiqi. 15-lipoxygenase-1 suppresses linoleic acid promotion of colorectal tumorigenesis through oxidative metabolism of PI3P_linoleic acid to inhibit membranous LRP5 recycling and WNT/β-catenin signaling activation [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2557.

BACKGROUND & AIMS: The peroxisome proliferator activated receptor delta (PPARD) regulates cell metabolism, proliferation, and inflammation and has been associated with gastric and other cancers. Villin-positive epithelial cells are a small population of quiescent gastric progenitor cells. We expressed PPARD from a villin promoter to investigate the role of these cells and PPARD in development of gastric cancer. METHODS: We analyzed gastric tissues from mice that express the Ppard (PPARD1 and PPARD2 mice) from a villin promoter, and mice that did not carry this transgene (controls), by histology and immunohistochemistry. We performed cell lineage tracing experiments and analyzed the microbiomes, chemokine and cytokine production, and immune cells and transcriptomes of stomachs of these mice. We also performed immunohistochemical analysis of PPARD levels in in 2 sets of human gastric tissue microarrays. RESULTS: Thirty-eight percent of PPARD mice developed spontaneous, invasive gastric adenocarcinomas, with severe chronic inflammation. Levels of PPARD were increased in human gastric cancer tissues, compared with non-tumor tissues, and associated with gastric cancer stage and grade. We found an inverse correlation between level of PPARD in tumor tissue and patients survival time. Gastric microbiomes from PPARD and control mice did not differ significantly. Lineage-tracing experiments identified villin-expressing gastric progenitor cells (VGPCs) as the origin of gastric tumors in PPARD mice. In these mice, PPARD upregulated CCL20 and CXCL1, which increased infiltration of the gastric mucosa by immune cells. Immune cell production of inflammatory cytokines promoted chronic gastric inflammation and expansion and transformation of VGPCs, leading to tumorigenesis. We identified a positive-feedback loop between PPARD and interferon gamma signaling that sustained gastric inflammation to induce VGPC transformation and gastric carcinogenesis. CONCLUSIONS: We found PPARD overexpression in VPGCs to result in inflammation, dysplasia, and tumor formation. PPARD and VGPCs might be therapeutic targets for stomach cancer.

Colorectal carcinogenesis (CRC) progression requires additional molecular mechanisms to APC mutations/aberrant β-catenin signaling. PPARD is a druggable ligand-activated nuclear receptor that regulates essential genes involved in cell fate. PPARD is upregulated in intestinal epithelial cells (IECs) of human colorectal adenomas and adenocarcinomas. The mechanistic significance of PPARD upregulation in CRC remains unknown. Here we show that targeted PPARD overexpression in IECs of mice strongly augmented β-catenin activation via BMP7/TAK1 signaling, promoted intestinal tumorigenesis in Apcmin mice, and accelerated CRC progression and invasiveness in mice with IEC-targeted ApcΔ580 mutation. Human CRC invasive fronts had higher PPARD expression than their paired adenomas. A PPARD agonist (GW501516) enhanced APCΔ580 mutation-driven CRC, while a PPARD antagonist (GSK3787) suppressed it. Functional proteomics analyses and subsequent validation studies uncovered PPARD upregulation of multiple pro-invasive pathways that drive CRC progression (e.g. PDGFRβ, AKT1, CDK1 and EIF4G1). Our results identify novel mechanisms by which PPARD promotes CRC invasiveness and provide the rational for the development of PPARD antagonists to suppress CRC.

Little is known about the cell origin of gastric cancer. Peroxisome proliferator-activated receptor-delta (PPARD) is a druggable ligand-activated nuclear receptor that impacts protumorigenic cellular events. However, PPARD’s role in tumorigenesis, especially gastric tumorigenesis, remains to be defined. We found that targeting PPARD overexpression in murine gastric progenitor cells (GPC), via a villin promoter, spontaneously induced gastric tumorigenesis that progressed to invasive adenocarcinoma. PPARD overexpression in GPC upregulated tumorigenic proinflammatory cytokine and CD44 expression, expanded GPC populationin vivo, enhanced GPC self-renewal and proliferation in organoid cultures, and endowed these cells with tumorigenic properties. Our findings identify PPARD as a driver of gastric tumorigenesis via GPC transformation.

The reception and integration of the plethora of signals a cell receives from its microenvironment determines the cell's 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. Significance Mounting evidence indicates that cancer stem cells (CSCs) are mainly responsible for cancer aggressiveness, drug resistance, and tumor relapse. CD44, especially CD44v isoforms, have been identified as CSC surface markers for isolating and enriching CSCs in different types of cancers. The current findings concerning the critical role of CD44/CD44v in regulation of cancer stemness and the research status of CD44/CD44v as biomarkers and therapeutic targets in cancer are summarized. The current challenges and future directions that may lead to best use of CD44/CD44v for clinical applications are also discussed.

Solid tumors are composed of mutually interacting cancer cells and tumor microenvironment. Many environmental components, such as extracellular matrix (ECM), mesenchymal stem cells, endothelial and immune cells, and various growth factors and cytokines, provide signals, either stimulatory or inhibitory, to cancer cells and determine their fates. Meanwhile, cancer cells can also educate surrounding cells or tissues to undergo changes that are in favorable of tumor progression. CD44, as a transmembrane receptor for hyaluronic acid (HA) and many other ECM components and a coreceptor for growth factors and cytokines, is a critical cell surface molecule that can sense, integrate, and transduce cellular microenvironmental signals to membrane-associated cytoskeletal proteins or to cell nucleus to regulate a variety of gene expressions that govern cell behaviors. Mounting evidence suggests that CD44, particularly CD44v isoforms, are cancer stem cell (CSC) markers and critical regulators of cancer stemness, including self-renewal, tumor initiation, and metastasis. Thus, CD44 is widely used alone or in combination with other cell surface markers to isolate or enrich CSCs through fluorescence-activated cell sorting of dissociated single cells that originate from the patient, xenograft tumor tissues, or tumor cell cultures. Sorted cells are cultured in a specialized culture medium for spheroid formation or inoculated into immunodeficient mice for the analysis of tumorigenic or metastatic potential. In this chapter, detailed experimental methods regarding CD44

Solid tumors are composed of mutually interacting cancer cells and tumor microenvironment. Many environmental components, such as extracellular matrix (ECM), mesenchymal stem cells, endothelial and immune cells, and various growth factors and cytokines, provide signals, either stimulatory or inhibitory, to cancer cells and determine their fates. Meanwhile, cancer cells can also educate surrounding cells or tissues to undergo changes that are in favorable of tumor progression. CD44, as a transmembrane receptor for hyaluronic acid (HA) and many other ECM components and a coreceptor for growth factors and cytokines, is a critical cell surface molecule that can sense, integrate, and transduce cellular microenvironmental signals to membrane-associated cytoskeletal proteins or to cell nucleus to regulate a variety of gene expressions that govern cell behaviors. Mounting evidence suggests that CD44, particularly CD44v isoforms, are cancer stem cell (CSC) markers and critical regulators of cancer stemness, including self-renewal, tumor initiation, and metastasis. Thus, CD44 is widely used alone or in combination with other cell surface markers to isolate or enrich CSCs through fluorescence-activated cell sorting of dissociated single cells that originate from the patient, xenograft tumor tissues, or tumor cell cultures. Sorted cells are cultured in a specialized culture medium for spheroid formation or inoculated into immunodeficient mice for the analysis of tumorigenic or metastatic potential. In this chapter, detailed experimental methods regarding CD44+ tumor cell isolation, spheroid culture, and characterization will be described.

Purpose: The dismal prognosis of pancreatic cancer has been linked to poor tumor differentiation. However, molecular basis of pancreatic cancer differentiation and potential therapeutic value of the underlying molecules remain unknown. We investigated the mechanistic underexpression of Krüppel-like factor 4 (KLF4) in pancreatic cancer and defined a novel epigenetic pathway of its activation for pancreatic cancer differentiation and treatment. Experimental Design: Expressions of KLF4 and DNMT1 in pancreatic cancer tissues were determined by IHC and the genetic and epigenetic alterations of KLF4 in and KLF4′s impact on differentiation of pancreatic cancer were examined using molecular biology techniques. The function of dietary 3,3′-diindolylmethane (DIM) on miR-152/DNMT1/KLF4 signaling in pancreatic cancer was evaluated using both cell culture and animal models. Results: Overexpression of DNMT1 and promoter hypermethylation contributed to decreased KLF4 expression in and associated with poor differentiation of pancreatic cancer. Manipulation of KLF4 expression significantly affected differentiation marker expressions in pancreatic cancer cells. DIM treatment significantly induced miR-152 expression, which blocked DNMT1 protein expression and its binding to KLF4 promoter region, and consequently reduced promoter DNA methylation and activated KLF4 expression in pancreatic cancer cells. In addition, DIM treatment caused significant inhibition of cell growth in vitro and tumorigenesis in animal models of pancreatic cancer. Conclusions: This is the first demonstration that dysregulated KLF4 expression associates with poor differentiation of pancreatic cancer. Epigenetic activation of miR-152/DNMT1/KLF4 signaling pathway by dietary DIM causes differentiation and significant growth inhibition of pancreatic cancer cells, highlighting its translational implications for pancreatic and other cancers. Clin Cancer Res; 23(18); 5585–97. ©2017 AACR.

Peroxisome proliferator-activated receptor-δ (PPARD) is upregulated in many major human cancers, but the role that its expression in cancer cells has in metastasis remains poorly understood. Here, we show that specific PPARD downregulation or genetic deletion of PPARD in cancer cells significantly repressed metastasis in various cancer models in vivo. Mechanistically, PPARD promoted angiogenesis via interleukin 8 in vivo and in vitro. Analysis of transcriptome profiling of HCT116 colon cancer cells with or without genetic deletion of PPARD and gene expression patterns in The Cancer Genome Atlas colorectal adenocarcinoma database identified novel pro-metastatic genes (GJA1, VIM, SPARC, STC1, SNCG) as PPARD targets. PPARD expression in cancer cells drastically affected epithelial-mesenchymal transition, migration, and invasion, further underscoring its necessity for metastasis. Clinically, high PPARD expression in various major human cancers (e.g., colorectal, lung, breast) was associated with significantly reduced metastasis-free survival. Our results demonstrate that PPARD, a druggable protein, is an important molecular target in metastatic cancer.

The expression of 15-lipoxygenase-1 (15-LOX-1) is downregulated in colon cancer and other major cancers, and 15-LOX-1 reexpression in cancer cells suppresses colonic tumorigenesis. Various lines of evidence indicate that 15-LOX-1 expression suppresses premetastatic stages of colonic tumorigenesis; nevertheless, the role of 15-LOX-1 loss of expression in cancer epithelial cells in metastases continues to be debated. Hypoxia, a common feature of the cancer microenvironment, promotes prometastatic mechanisms such as the upregulation of hypoxia-inducible factor (HIF)-1α, a transcriptional master regulator that enhances cancer cell metastatic potential, angiogenesis, and tumor cell invasion and migration. We have, therefore, tested whether restoring 15-LOX-1 in colon cancer cells affects cancer cells' hypoxia response that promotes metastasis. We found that 15-LOX-1 reexpression in HCT116, HT29LMM, and LoVo colon cancer cells inhibited survival, vascular endothelial growth factor (VEGF) expression, angiogenesis, cancer cell migration and invasion, and HIF-1α protein expression and stability under hypoxia. These findings demonstrate that 15-LOX-1 expression loss in cancer cells promotes metastasis and that therapeutically targeting ubiquitous 15-LOX-1 loss in cancer cells has the potential to suppress metastasis.