W Xu

15-Lipoxygenase-1 suppression of colitis-associated colon cancer through inhibition of the IL-6/STAT3 signaling pathway

The IL‐6/signal transducer and activator of transcription 3 (STAT3) pathway is a critical signaling pathway for colitis‐associated colorectal cancer (CAC). Peroxisome proliferator‐activated receptor (PPAR)‐δ, a lipid nuclear receptor, up‐regulates IL‐6. 15‐Lipoxygenase‐1 (15‐LOX‐1), which is crucial to production of lipid signaling mediators to terminate inflammation, down‐regulates PPAR‐δ. 15‐LOX‐1 effects on IL‐6/STAT3 signaling and CAC tumorigenesis have not been determined. We report that intestinally targeted transgenic 15‐LOX‐1 expression in mice inhibited azoxymethane‐ and dextran sodium sulfate‐induced CAC, IL‐6 expression, STAT3 phosphorylation, and IL‐6/STAT3 downstream target (Notch3 and MUC1) expression. 15‐LOX‐1 down‐regulation was associated with IL‐6 up‐regulation in human colon cancer mucosa. Reexpression of 15‐LOX‐1 in human colon cancer cells suppressed IL‐6 mRNA expression, STAT3 phosphorylation, IL‐6 promoter activity, and PPAR‐δ mRNA and protein expression. PPAR‐δ overexpression in colonic epithelial cells promoted CAC tumorigenesis in mice and increased IL‐6 expression and STAT3 phosphorylation, whereas concomitant 15‐LOX‐1 expression in colonic epithelial cells (15‐LOX‐1‐PPAR‐δ‐Gut mice) suppressed these effects: the number of tumors per mouse (mean ± sem) was 4.22 ± 0.68 in wild‐type littermates, 6.67 ± 0.83 in PPAR‐δ‐Gut mice (P= 0.026), and 2.25 ± 0.25 in 15‐LOX‐1‐PPAR‐δ‐Gut mice (P = 0.0006). Identification of 15‐LOX‐1 suppression of PPAR‐δ to inhibit IL‐6/STAT3 signaling‐driven CAC tumorigenesis provides mechanistic insights that can be used to molecularly target CAC.—Mao, F., Xu, M., Zuo, X., Yu, J., Xu, W., Moussalli, M. J., Elias, E., Li, H. S., Watowich, S. S., Shureiqi, I. 15‐Lipoxygenase‐1 suppression of colitis‐associated colon cancer through inhibition of the IL‐6/STAT3 signaling pathway. FASEB J. 29, 2359‐2370 (2015). www.fasebj.org

Metastasis regulation by PPARD expression in cancer cells

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.

Abstract PD03-06: Simvastatin radiosensitizes differentiated and stem-like breast cancer cell lines and is associated with improved local control in inflammatory breast cancer patients treated with post-mastectomy radiation

Among women with non-inflammatory triple-negative and triple-negative inflammatory breast cancer (IBC), the 5-yr actuarial rates of local failure after radiation are 11%–35% and 45%, respectively, in part influenced by the contribution of radioresistant cancer stem cells to these cancers. Herein we explored the radiosensitization of breast cancer stem-like cells in vitro and examined the influence on local control after post-mastectomy radiation (PMRT) among IBC patients taking statins. SUM149, SUM159 and MCF-7 cells were cultured in standard monolayer cultures and stem cell enriching anchorage independent clonogenic cultures with simvastatin and treated with increasing concentrations of radiation. Survival curves were generated and t-test was used to compare surviving fraction (SF) of groups. p Simvastatin radiosensitized all cell lines in both types of clonogenic culture assays. The triple-negative IBC cell line SUM149 had the greatest response to combined treatment regardless of the radiation dose used in monolayer cultures (SF2: 0.417 vs 0.319, SF4: 0.136 vs 0.075, SF6: 0.026 vs 0.018, in control vs treated respectively, all p Patients with IBC and triple negative non-IBC breast cancer have the highest rates of local failure and no available known radiosensitizers. Here we report significant improvement in local control after PMRT among statin users with IBC and significant radiosensitization across triple-negative and IBC cell lines of multiple subtypes using simvastatin. Clinical value in patients without hypercholesterolemia remains to be established. These encouraging data suggest simvastatin may be an appropriate radiosensitizing agent for clinical trials. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr PD03-06.

Novel inflammatory breast cancer cell line, MDA-IBC-1 expresses and secretes WISP3, a putative tumor suppressor in inflammatory breast cancer.

CTRC-AACR San Antonio Breast Cancer Symposium: 2008 Abstracts Abstract #1051 Wnt-1-induced secreted protein 3 (WISP3) is a member of the CCN family of proteins, which also includes connective tissue growth factor (CTGF), Cy61, Nov, WISP1, and WISP2. WISP3, as well as the other CCN family members, plays important roles in cellular processes such as proliferation, migration, and survival. WISP3 was originally isolated as Lost in Inflammatory Breast Cancer (LIBC) based on its expression in only 20% of human IBC tumors. Lack of WISP3 expression has also been reported in the IBC-derived cell line SUM-149. In SUM149 cells WISP3 has been reported to function as a tumor suppressor capable of decreasing proliferation and maintaining an epithelial phenotype. Here we examine the expression of WISP3 in a novel aggressive IBC cell line with epithelial-mesenchymal transition (EMT) morphology and abundant WISP3 expression. MDA-IBC-1 cells were isolated from the pleural fluid of a patient with ER+, PR-, HER2/neu- IBC. In 2-dimensional (2D) adherent culture, MDA-IBC-1 cells exhibit fibroblast-like EMT morphology and express EMT-associated proteins such as N-cadherin, vimentin, and fibronectin. Correspondingly, E-cadherin protein expression is absent in 2D cultures. MDA-IBC-1 cells derived from 2D cultures form mammospheres when cultured in 3-dimensional (3D) progenitor promoting conditions. Interestingly, the expression of EMT molecular markers is absent in 3D progenitor promoting mammosphere culture. MDA-IBC-1 cells exhibit robust WISP3 protein expression in both 2D and 3D culture (1.8-fold higher in 3D). In addition, MDA-IBC-1 cells secrete WISP3 as indicated by the presence of WISP3 protein in MDA-IBC-1 derived conditioned media. WISP3 binding is reported to decrease insulin-like growth factor-1 (IGF-1) activation of the IGF1R signaling cascade in SUM-149 cells. IGFR1 protein is present in 2D and 3D cultured MDA-IBC-1 cells. IGF-1 protein overlay assays using conditioned media demonstrate a WISP3:IGF-1 interaction, suggesting functional folding. Serum-free MDA-IBC-1 conditioned media did not increase basal SUM149 proliferation and it inhibited IGF-1 stimulated proliferation to serum-free (basal) levels (p≤0.05). In addition, culturing SUM-149 cells in conditioned media obtained from IBC-1 cells significantly alters SUM-149 to a completely fibroblastic morphology. These data demonstrate that MDA-IBC-1 cells represent an IBC cell line with novel expression and regulation of WISP3. MDA-IBC-1 cells may better represent the cohort of IBC patients whose tumors express WISP3. This data suggest WISP3 may not function as a tumor suppressor in all IBC patients. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 1051.

Populations of cells exhibiting epithelial-mesenchymal transition (EMT) or stem cell characteristics can be propagated from a single inflammatory breast cancer (IBC) cell line based on culture conditions.

Abstract #103 We have previously reported the isolation of a novel IBC cell line (MDA-IBC-1) from human pleural effusion fluid by passaging cells under 3-dimensional (3D) mammosphere (progenitor-promoting) conditions and isolating attached 2-dimensional (2D) cells from ultralow attachment plates. Mammosphere culture is serum-free and growth factor enriched. Serum-starving 2D cells leads to spontaneous formation of floating mammospheres with the same percentage of CD44+24- cells as 3D cultured mammospheres. 2D cells display distinct fibroblastic morphology. Here we report that passage in 2D (standard) or 3D (mammosphere) culture selects for different molecular phenotypes: EMT in 2D and stem/progenitor-like in 3D. 3D cells are generated from 2D cells for all experiments and are examined at second passage. RT-PCR reveals embryonic stem cell pluripotency factors Oct-4, Nanog, Sox-2 and Rex-1 are expressed in 3D but not 2D MDA-IBC-1 cells. MicroRNAs reported to be involved in self-renewal of breast cancer cells including Let-7a,b,c,d,e and f are decreased in 3D cultures (>5 fold decrease) compared to 2D. MiR16, 21, 125b and 200c are also markedly reduced in 3D vs. 2D cells. On the other hand, EMT proteins, N-cadherin, vimentin, and fibronectin are only expressed in 2D culture and higher expression of snail and slug is observed in 2D compared to 3D. Correspondingly, E-cadherin is absent from 2D cells. Moreover, Notch1, a putative stem cell signaling pathway protein that has been shown to promote EMT by snail induction is upregulated in 2D cells compared to 3D, and in response to radiation in both 2D and 3D. It has been reported in some systems that EMT promotion can induce stem cells. The data herein suggest the MDA-IBC-1 maintains pluripotent cells capable of both EMT and stem-like phenotypes. MDA-IBC-1 provides a unique model to examine the interaction between stem cells and EMT and to examine the mechanisms of resistance and proliferation of both cell types. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 103.

PPARD regulation in gastric progenitor cells drives gastric tumorigenesis in mice

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 population in 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.

Abstract 1959: 15-lipoxygenase-1 suppression of colitis-associated colon cancer through inhibition of the IL-6/STAT3 signaling pathway

The IL-6/STAT3 pathway is a critical signaling pathway for colitis-associated colorectal cancer (CAC). PPAR-delta (PPAR-d), a lipid nuclear receptor, upregulates IL-6. 15-Lipoxygenase-1 (15-LOX-1) is crucial to produce lipid signaling mediators to terminate inflammation. 15-LOX-1 downregulates PPAR-d. 15-LOX-1 effects on IL-6/STAT3 signaling and CAC tumorigenesis are unknown. We here report that intestinally targeted transgenic 15-LOX-1 expression in mice (15-LOX-1-Gut mice) inhibited azoxymethane and dextran sodium sulfate-induced CAC, IL-6 expression, STAT3 phosphorylation, and IL-6/STAT3 downstream target expression (Notch and MUC1). 15-LOX-1 downregulation was associated with IL-6 upregulation in human colon cancer mucosa. Re-expression of 15-LOX-1 in human colon cancer cells suppressed IL-6 mRNA expression, STAT3 phosphorylation, IL-6 promoter activity, and PPAR-d mRNA and protein expression. PPAR-d overexpression in colonic epithelial cells (PPAR-d-Gut mice) promoted CAC tumorigenesis in mice and increased IL-6 expression and STAT3 phosphorylation, whereas concomitant 15-LOX-1 expression in colonic epithelial cells (15-LOX-1-PPAR-d-Gut mice) suppressed these effects [e.g., tumor number per mouse (mean ± SE) was 4.22 ± 0.68 in wild-type littermates, 6.67 ± 0.83 in PPAR-d-Gut mice (p = 0.026), and 2.25 ± 0.25 in 15-LOX-1-PPAR-d-Gut mice (p = 0.0006)]. Identification of 15-LOX-1 suppression of PPAR-d to inhibit IL-6/STAT3 signaling-driven CAC tumorigenesis provides mechanistic insights that can be utilized to molecularly target CAC. Citation Format: Xiangsheng Zuo, Fei Mao, Min Xu, Weiguo Xu, Rui Tian, Micheline J. Micheline J. Moussalli, Elias Elias, Haiyan S. Li, Stephanie S. Watowich, Imad Shureiqi. 15-lipoxygenase-1 suppression of colitis-associated colon cancer through inhibition of the IL-6/STAT3 signaling pathway. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1959. doi:10.1158/1538-7445.AM2015-1959

Abstract P1-06-05: Expansion of tumor initiating cells is mediated by tumor microenvironment in breast cancer metastasis

Background: Breast cancer metastasis which ultimately results in breast cancer death, is an event believed to be initiated by the migration of tumor initiating cells (TIC) from the primary tumor to niches for micrometastatic disease. Recent data suggests the tumor microenvironment promotes TIC. The clinical relevance of secreted factors from the microenvironment on TIC surrogate, mammosphere (MS) formation and MS sensitivity to drug therapy was investigated using breast cancer patient fluids inherently conditioned by the tumor microenvironment: post-operative seromas and malignant pleural effusions. Methods: Fluids from 48 patients with breast cancer (15 seromas and 33 pleural effusions) and mesenchymal stem cells (MSC) from healthy donors were collected on IRB approved protocols. Cellular components were eliminated from patient-derived fluids using density-gradient centrifugation. MSC conditioned media (MSC-CM) was collected from 3D cultures of primary MSC. Luminex multiplex array platform was used to characterize 79 cytokine and growth factor components of all fluids. In addition, MSC-CM and patient-derived fluids were added to cultures of breast cancer cell lines: MCF-7, an estrogen receptor (ER)-positive cell line; SUM149, a triple-negative inflammatory breast cancer cell line; and SUM159, a triple-negative metaplastic breast cancer cell line and MS forming efficiency was examined. Results: Our results show that pleural effusions and seromas are enriched for factors also secreted by MSC such as MCP-1, GRO, IL-6, and VEGF-A. We found remarkable similarities regarding the cytokines and growth factors profile in pleural effusions and seromas. Both patient-derived fluids have comparable amount of Angiopoetin-2, Leptin, TNF-beta, VEGF, IL-2, IL-3, IL-4 and IL-10. EGF, TNF-alpha, IL-1, IL-6, IL-8 and IL-16 were significantly different between pleural effusions and seromas. Seroma fluid from bilateral drains in a patient with an invasive cancer and a contralateral benign mastectomy had very similar cytokine concentrations. Moreover, MSC-CM and pleural fluids from ER-positive and ER-negative patients increased the MS formation efficiency of both triple-negative cell lines while seroma fluids from ER-positive and ER-negative patients increased the MS formation efficiency of ER-positive cell line MCF-7. Finally, we evaluated the impact of a panel of drugs (simvastatin, pravastatin and erlotinib) on cell cultures grown with MSC-CM and patient-derived fluids. We found that the effect of chemotherapies on MS formation can be attenuated by patient-derived fluids. Conclusions: Seroma and pleural effusion fluids from breast cancer patients have similar cytokine profiles, change MS formation efficiency of standard breast cancer cell models, and mediate sensitivity to therapy. Here we demonstrate that host and microenvironmental factors are critical for determining resistance to therapy and may be independent of obvious tumor related factors. Future studies will investigate the prognostic implications of factors that promote TIC survival in the fluid tumor microenvironment. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P1-06-05.

Abstract P5-03-05: Histone deacetylase (HDAC)-inhibitor mediated reprogramming drives cancer cells to the pentose phosphate metabolic pathway

Recent studies have shown that energy metabolism in human pluripotent cells contrasts sharply with energy metabolism in differentiated cell types. Specifically, it has been shown that nuclear reprogramming from somatic cells to induced pluripotent stem cells is associated with a switch from oxidative to glycolytic metabolism. Whether a metabolic switch also occurs in reprogrammed/dedifferentiated breast cancer cells is unknown. Moreover, the function of the metabolic state in stemness is poorly understood and no data are available on whether breast cancer stem cells (CSCs) are metabolically different from committed cancer cells. Herein we demonstrated that HDAC inhibitors reprogram committed single aldefluor negative breast cancer cells into aldefluor positive cells (10.3 ± 2.8 vs 21.3 ±3.7% untreated vs treated P Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P5-03-05.

P1-04-01: The Mechanism of Anti-Breast Cancer TICs Effect of Pyrvinium Pamoate Is through WNT/beta-Catenin Signaling.

We have previously shown that pyrvinium pamoate can decrease breast cancer TICs in vitro and shrink the tumor size in vivo. Although pyrvinium pamoate has been shown to target beta-catenin through activating CK-1alpha in a vitro model, the mechanism of its anti-breast cancer TICs effect is unknown. Herein, we use a constitutively active WNT/beta-catenin signaling construct EBETAP (ref) to determine if the anti-breast TIC effect of pyrvinium pamoate is through WNT/beta-catenin signaling. Using aldefluor expression and mammosphere formation efficiency as TIC surrogate assays, we found that TICs of SUM-159 transfected with EBETAP construct are resistant to pyrvinium pamoate treatment compared to control cells. Moreover, microarray analysis reveals a series of genes and signaling downstream of WNT-catenin were down-regulated in SUM-159 cells treated with pyrvinium pamoate. In summary, mechanism of anti-breast cancer TICs effect of pyrvinium pamoate is through WNT/beta-catenin signaling. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P1-04-01.